Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Disruption of androgen regulation in the prostate by the environmental contaminant hexachlorobenzene

Hexachlorobenzene (HCB) is a persistent environmental contaminant that has the potential to interfere with steroid hormone regulation. The prostate requires precise control by androgens to regulate its growth and function. To determine if HCB impacts androgen action in the prostate, we used a number of methods. Our in vitro cell-culture-based assay used a firefly luciferase reporter gene driven by an androgen-responsive promoter. In the presence of dihydrotestosterone, low concentrations (0.5-5 nM) of HCB increased the androgen-responsive production of firefly luciferase and high concentrations of HCB (> 10 microM) suppressed this transcriptional activity. Results from a binding assay showed no evidence of affinity between HCB and the androgen receptor. We also tested HCB for in vivo effects using transgenic mice in which the transgene was a prostate-specific, androgen-responsive promoter upstream of a chloramphenicol acetyl transferase (CAT) reporter gene. In 4-week-old mice, the proportion of dilated prostate acini, a marker of sexual maturity, increased in the low HCB dose group and decreased in the high HCB dose mice. In the 8-week-old mice, there was a significant decrease in both CAT activity and prostate weight upon exposure to 20 mg/kg/day HCB. Therefore, in vitro and in vivo data suggest that HCB weakly agonizes androgen action, and consequently, low levels of HCB enhanced androgen action but high levels of HCB interfered. Environmental contaminants have been implicated in the rising incidence of prostate cancer, and insight into the mechanisms of endocrine disruption will help to clarify their role.

The synthesis of novel nitroxides and their application as small-molecule antioxidants and profluorescent probes for oxidative stress

The detection and potential treatment of oxidative stress in biological systems has been explored using isoindoline-based nitroxide radicals. A novel tetraethyl-fluorescein nitroxide was synthesised for its use as a profluorescent probe for redox processes in biological systems. This tetraethyl system, as well as a tetramethyl-fluorescein nitroxide, were shown to be sensitive and selective probes for superoxide in vitro. The redox environment of cellular systems was also explored using the tetramethylfluorescein species based on its reduction to the hydroxylamine. Flow cytometry was employed to assess the extent of nitroxide reduction, reflecting the overall cellular redox environment. Treatment of normal fibroblasts with rotenone and 2-deoxyglucose resulted in an oxidising cellular environment as shown by the lack of reduction of the fluorescein-nitroxide system. Assessment of the tetraethyl-fluorescein nitroxide system in the same way demonstrated its enhanced resistance to reduction and offers the potential to detect and image biologically relevant reactive oxygen species directly. Importantly, these profluorescent nitroxide compounds were shown to be more effective than the more widely used and commercially available probes for reactive oxygen species such as 2’,7’-dichlorodihydrofluorescein diacetate. Fluorescence imaging of the tetramethyl-fluorescein nitroxide and a number of other rhodamine-nitroxide derivatives was undertaken, revealing the differential cellular localisation of these systems and thus their potential for the detection of redox changes in specific cellular compartments. As well as developing novel methods for the detection of oxidative stress, a number of novel isoindoline nitroxides were synthesised for their potential application as small-molecule antioxidants. These compounds incorporated known pharmacophores into the isoindoline-nitroxide structure in an attempt to increase their efficacy in biological systems. A primary and a secondary amine nitroxide were synthesised which incorporated the phenethylamine backbone of the sympathomimetic amine class of drugs. Initial assessment of the novel primary amine derivative indicated a protective effect comparable to that of 5-carboxy-1,1,3,3- tetramethylisoindolin-2-yloxyl. Methoxy-substituted nitroxides were also synthesised as potential antioxidants for their structural similarity to some amphetamine type stimulants. A copper-catalysed methodology provided access to both the mono- and di-substituted methoxy-nitroxides. Deprotection of the ethers in these compounds using boron tribromide successfully produced a phenolnitroxide, however the catechol moiety in the disubstituted derivative appeared to undergo reaction with the nitroxide to produce quinone-like degradation products. A novel fluoran-nitroxide was also synthesised from the methoxy-substituted nitroxide, providing a pH-sensitive spin probe. An amino-acid precursor containing a nitroxide moiety was also synthesised for its application as a dual-action antioxidant. N-Acetyl protection of the nitroxide radical was necessary prior to the Erlenmeyer reaction with N-acetyl glycine. Hydrolysis and reduction of the azlactone intermediate produced a novel amino acid precursor with significant potential as an effective antioxidant.

Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities*

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-b-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsinlike activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin- like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyland radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly- Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3,4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by twodimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities

Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

Background: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods. HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (∼1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/g Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice. © 2011 Valley-Omar et al; licensee BioMed Central Ltd.

Ionic liquid pre-treatment and fractionation of sugarcane bagasse for the production of bioethanol

Pretretament is an essential and expensive processing step for the manufacturing of ethanol from lignocellulosic raw materials. Ionic liquids are a new class of solvents that have the potential to be used as pretreatment agents. The attractive characteristics of ionic liquid pretreatment of lignocellulosics such as thermal stability, dissolution properties, fractionation potential, cellulose decrystallisation capacity and saccharification impact are investigated in this thesis. Dissolution of bagasse with 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) at high temperatures (110 �‹C to 160 �‹C) is investigated as a pretreatment process. Material balances are reported and used along with enzymatic saccharification data to identify optimum pretreatment conditions (150 �‹C for 90 min). At these conditions, the dissolved and reprecipitated material is enriched in cellulose, has a low crystallinity and the cellulose component is efficiently hydrolysed (93 %, 3 h, 15 FPU). At pretreatment temperatures < 150 �‹C, the undissolved material has only slightly lower crystallinity than the starting. At pretreatment temperatures . 150 �‹C, the undissolved material has low crystallinity and when combined with the dissolved material has a saccharification rate and extent similar to completely dissolved material (100 %, 3h, 15 FPU). Complete dissolution is not necessary to maximize saccharification efficiency at temperatures . 150 �‹C. Fermentation of [C4mim]Cl-pretreated, enzyme-saccharified bagasse to ethanol is successfully conducted (85 % molar glucose-to-ethanol conversion efficiency). As compared to standard dilute acid pretreatment, the optimised [C4mim]Cl pretreatment achieves substantially higher ethanol yields (79 % cf. 52 %) in less than half the processing time (pretreatment, saccharification, fermentation). Fractionation of bagasse partially dissolved in [C4mim]Cl to a polysaccharide rich and a lignin rich fraction is attempted using aqueous biphasic systems (ABSs) and single phase systems with preferential precipitation. ABSs of ILs and concentrated aqueous inorganic salt solutions are achievable (e.g. [C4mim]Cl with 200 g L-1 NaOH), albeit they exhibit a number of technical problems including phase convergence (which increases with increasing biomass loading) and deprotonation of imidazolium ILs (5 % - 8 % mol). Single phase fractionation systems comprising lignin solvents / cellulose antisolvents, viz. NaOH (2M) and acetone in water (1:1, volume basis), afford solids with, respectively, 40 % mass and 29 % mass less lignin than water precipitated solids. However, this delignification imparts little increase in saccharification rates and extents of these solids. An alternative single phase fractionation system is achieved simply by using water as an antisolvent. Regulating the water : IL ratio results in a solution that precipitates cellulose and maintains lignin in solution (0.5 water : IL mass ratio) in both [C4mim]Cl and 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc)). This water based fractionation is applied in three IL pretreatments on bagasse ([C4mim]Cl, 1-ethyl-3-methyl imidazolium chloride ([C2mim]Cl) and [C2mim]OAc). Lignin removal of 10 %, 50 % and 60 % mass respectively is achieved although only 0.3 %, 1.5 % and 11.7 % is recoverable even after ample water addition (3.5 water : IL mass ratio) and acidification (pH . 1). In addition the recovered lignin fraction contains 70 % mass hemicelluloses. The delignified, cellulose-rich bagasse recovered from these three ILs is exposed to enzyme saccharification. The saccharification (24 h, 15 FPU) of the cellulose mass in starting bagasse, achieved by these pretreatments rank as: [C2mim]OAc (83 %)>>[C2mim]Cl (53 %)=[C4mim]Cl(53%). Mass balance determinations accounted for 97 % of starting bagasse mass for the [C4mim]Cl pretreatment , 81 % for [C2mim]Cl and 79 %for [C2mim]OAc. For all three IL treatments, the remaining bagasse mass (not accounted for by mass balance determinations) is mainly (more than half) lignin that is not recoverable from the liquid fraction. After pretreatment, 100 % mass of both ions of all three ILs were recovered in the liquid fraction. Compositional characteristics of [C2mim]OAc treated solids such as low lignin, low acetyl group content and preservation of arabinosyl groups are opposite to those of chloride IL treated solids. The former biomass characteristics resemble those imparted by aqueous alkali pretreatment while the latter resemble those of aqueous acid pretreatments. The 100 % mass recovery of cellulose in [C2mim]OAc as opposed to 53 % mass recovery in [C2mim]Cl further demonstrates this since the cellulose glycosidic bonds are protected under alkali conditions. The alkyl chain length decrease in the imidazolium cation of these ILs imparts higher rates of dissolution and losses, and increases the severity of the treatment without changing the chemistry involved.

Metabolite profiling and pathway analysis of acute hepatitis rats by UPLC-ESI MS combined with pattern recognition methods

BACKGROUND & AIMS Metabolomics is comprehensive analysis of low-molecular-weight endogenous metabolites in a biological sample. It could enable mapping of perturbations of early biochemical changes in diseases and hence provide an opportunity to develop predictive biomarkers that could provide valuable insights into the mechanisms of diseases. The aim of this study was to elucidate the changes in endogenous metabolites and to phenotype the metabolic profiling of d-galactosamine (GalN)-inducing acute hepatitis in rats by UPLC-ESI MS. METHODS The systemic biochemical actions of GalN administration (ip, 400 mg/kg) have been investigated in male wistar rats using conventional clinical chemistry, liver histopathology and metabolomic analysis of UPLC- ESI MS of urine. The urine was collected predose (-24 to 0 h) and 0-24, 24-48, 48-72, 72-96 h post-dose. Mass spectrometry of the urine was analysed visually and via conjunction with multivariate data analysis. RESULTS Results demonstrated that there was a time-dependent biochemical effect of GalN dosed on the levels of a range of low-molecular-weight metabolites in urine, which was correlated with developing phase of the GalN-inducing acute hepatitis. Urinary excretion of beta-hydroxybutanoic acid and citric acid was decreased following GalN dosing, whereas that of glycocholic acid, indole-3-acetic acid, sphinganine, n-acetyl-l-phenylalanine, cholic acid and creatinine excretion was increased, which suggests that several key metabolic pathways such as energy metabolism, lipid metabolism and amino acid metabolism were perturbed by GalN. CONCLUSION This metabolomic investigation demonstrates that this robust non-invasive tool offers insight into the metabolic states of diseases.

The reduced expression of the HADH2 protein causes X-linked mental retardation, choreoathetosis, and abnormal behavior

Recently, we defined a new syndromic form of X-linked mental retardation in a 4-generation family with a unique clinical phenotype characterized by mild mental retardation, choreoathetosis, and abnormal behavior (MRXS10). Linkage analysis in this family revealed a candidate region of 13.4 Mb between markers DXS1201 and DXS991 on Xp11; therefore, mutation analysis was performed by direct sequencing in most of the 135 annotated genes located in the region. The gene (HADH2) encoding L-3-hydroxyacyl-CoA dehydrogenase II displayed a sequence alteration (c.574 C-->A; p.R192R) in all patients and carrier females that was absent in unaffected male family members and could not be found in 2,500 control X chromosomes, including in those of 500 healthy males. The silent C-->A substitution is located in exon 5 and was shown by western blot to reduce the amount of HADH2 protein by 60%-70% in the patient. Quantitative in vivo and in vitro expression studies revealed a ratio of splicing transcript amounts different from those normally seen in controls. Apparently, the reduced expression of the wild-type fragment, which results in the decreased protein expression, rather than the increased amount of aberrant splicing fragments of the HADH2 gene, is pathogenic. Our data therefore strongly suggest that reduced expression of the HADH2 protein causes MRXS10, a phenotype different from that caused by 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, which is a neurodegenerative disorder caused by missense mutations in this multifunctional protein.

ortho-Bromo(propa-1,2-dien-1-yl)arenes : substrates for domino reactions

o-Bromo(propa-1,2-dien-1-yl)arenes exhibit novel and orthogonal reactivity under Pd catalysis in the presence of secondary amines to form enamines (concerted Pd insertion, intramolecular carbopalladation, and terminative Buchwald–Hartwig coupling) and of amides to form indoles (addition, Buchwald–Hartwig cyclization, and loss of the acetyl group). The substrates for these reactions can be accessed in a reliable and highly selective two-step process from 2-bromoaryl bromides.

The role of enoyl reductase genes in phloridzin biosynthesis in apple

Phloridzin is the predominant polyphenol in apple (Malus× domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.

A combined neutralization-reionization mass spectrometric and theoretical study of oxyallyl and other elusive [C3,H4,O] neutrals

Five different anionic [C3′H4′O]•- isomers, i.e. the radical anions of acrolein, acetyl carbene, formyl methyl carbene, methoxy vinylidene, and oxyallyl are generated in an ion beam mass spectrometer and subjected to neutralization-reionization (NR) mass spectrometric experiments including neutral and ion decomposition difference (NIDD) mass spectrometry; the latter allows for the examination of the neutrals' unimolecular reactivity. Further, the anionic, the singlet and triplet neutral, and the cationic [C3′H4′O] •-/0/•+ potentialenergy surfaces are calculated at the B3LYP/6-311++G(d,p) level of theory. For some species, notably the singlet state of oxyallyl, the theoretical treatment is complemented by G2, CASSCF, and MR-CI calculations. Theory and experiment are in good agreement in that at the neutral stage (i) acrolein does not react within the μsec timescale, (ii) acetyl and formyl methyl carbenes isomerize to methyl ketene, (iii) methoxy vinylidene rearranges to methoxy acetylene, (iv) singlet 1A1 oxyallyl undergoes ring closure to cyclopropanone, and (v) triplet 3B2 oxyallyl may have a lifetime sufficient to survive a NR experiment.

Atmospheric gas plasma–induced ROS production activates TNF-ASK1 pathway for the induction of melanoma cancer cell apoptosis

Atmospheric gas plasmas (AGPs) are able to selectively induce apoptosis in cancer cells, offering a promising alternative to conventional therapies that have unwanted side effects such as drug resistance and toxicity. However, the mechanism of AGP-induced cancer cell death is unknown. In this study, AGP is shown to up-regulate intracellular reactive oxygen species (ROS) levels and induce apoptosis in melanoma but not normal melanocyte cells. By screening genes involved in apoptosis, we identify tumor necrosis factor (TNF)-family members as the most differentially expressed cellular genes upon AGP treatment of melanoma cells. TNF receptor 1 (TNFR1) antagonist-neutralizing antibody specifically inhibits AGP-induced apoptosis signal, regulating apoptosis signal-regulating kinase 1 (ASK1) activity and subsequent ASK1-dependent apoptosis. Treatment of cells with intracellular ROS scavenger N-acetyl-l-cysteine also inhibits AGP-induced activation of ASK1, as well as apoptosis. Moreover, depletion of intracellular ASK1 reduces the level of AGP-induced oxidative stress and apoptosis. The evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.

Paradoxical enhancement of fear extinction memory and synaptic plasticity by inhibition of the histone acetyltransferase p300

It is well established that the coordinated regulation of activity-dependent gene expression by the histone acetyltransferase (HAT) family of transcriptional coactivators is crucial for the formation of contextual fear and spatial memory, and for hippocampal synaptic plasticity. However, no studies have examined the role of this epigenetic mechanism within the infralimbic prefrontal cortex (ILPFC), an area of the brain that is essential for the formation and consolidation of fear extinction memory. Here we report that a postextinction training infusion of a combined p300/CBP inhibitor (Lys-CoA-Tat), directly into the ILPFC, enhances fear extinction memory in mice. Our results also demonstrate that the HAT p300 is highly expressed within pyramidal neurons of the ILPFC and that the small-molecule p300-specific inhibitor (C646) infused into the ILPFC immediately after weak extinction training enhances the consolidation of fear extinction memory. C646 infused 6 h after extinction had no effect on fear extinction memory, nor did an immediate postextinction training infusion into the prelimbic prefrontal cortex. Consistent with the behavioral findings, inhibition of p300 activity within the ILPFC facilitated long-term potentiation (LTP) under stimulation conditions that do not evoke long-lasting LTP. These data suggest that one function of p300 activity within the ILPFC is to constrain synaptic plasticity, and that a reduction in the function of this HAT is required for the formation of fear extinction memory.

A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.