XTH acts at the microfibril-matrix interface during cell elongation

Sulphorhodamine-labelled oligosaccharides of xyloglucan are incorporated into the cell wall of Arabidopsis and tobacco roots, and of cultured Nicotiana tabacum cells by the transglucosylase (XET) action of XTHs. In the cell wall of diffusely growing cells, the subcellular pattern of XET action revealed a 'fibrillar' pattern, different from the xyloglucan localization. The fibrillar fluorescence pattern had no net orientation in spherical cultured cells. It changed to transverse to the long axis when the cells started to elongate, a feature mirroring the rearrangements of cortical microtubules and the accompanying cellulose deposition. Interference with the polymerization of microtubules and with cellulose deposition inhibited this strong and 'fibrillar'-organized XET-action, whereas interference with actin-polymerization only decreased the intensity of enzyme action. Epidermal cells of a mutant with reduced cellulose synthesis also had low XET action. Root hairs (tip-growing cells) exhibited high XET-action over all their length, but lacked the specific parallel pattern. In both diffuse- and tip-growing cell types extraction of the incorporated fluorescent xyloglucans by a xyloglucan-specific endoglucanase reduced the fluorescence, but the 'fibrillar' appearance in diffuse growing cells was not eliminated. These results show that XTHs act on the xyloglucans attached to cellulose microfibrils. After incorporation of the fluorescent oligosaccharides, the xyloglucans decorate the cellulose microfibrils and become inaccessible to hydrolytic enzymes

A Comparison of Maize Stalk Rot Occurrence in Bt and Non-Bt Hybrids

Stalk rots, caused by a complex of fungal species, are among the most widespread and destructive diseases of maize. Larvae of the European corn borer (ECB) (Ostrinia nubilalis) promote stalk rot development by creating entry points for fungi, serving as vectors of pathogens, and causing physiological stress that may predispose plants to stalk decay. Field experiments were conducted in 1998, 1999, and 2000 to determine whether the use of transgenic Bt hybrids expressing insecticidal proteins would influence stalk rot symptoms (pith disintegration, pith discoloration, and lodging). Five hybrids representing different Bt types (or “Bt events”) (176, BT11, MON810, DBT418, and CBH351) were paired with their near-isogenic, non-Bt counterparts and subjected to treatments of manual and natural infestation with ECB larvae. Manual infestation resulted in significantly more ECB tunneling than natural infestation in 1998 and 1999 and significantly more lodging in 1998. There were significant linear correlations between ECB injury and stalk rot symptoms in non-Bt hybrids in 1998 and 1999, but not in 2000. A standard foliar insecticide treatment for ECB did not significantly affect stalk rot symptoms. In 1998, Bt hybrids had significantly less ECB tunneling, stalk discoloration, pith disintegration, and lodging compared with non-Bt hybrids, but these effects depended upon the Bt event and the infestation treatment. Similar but less pronounced effects of Bt events were observed in 1999. The 2000 results were more variable; the amount of pith disintegration was significantly lower but discoloration was significantly higher in the BT11 hybrid compared with its non-Bt counterpart, and the amount of lodging was significantly higher in the event 176 hybrid compared with its non-Bt counterpart. The ratio of stalk strength to grain weight did not consistently differ between Bt and non-Bt hybrids. These results indicate that, although specific Bt events in some years may cause reductions in stalk rot, the overall effect of Bt transformation on stalk rot occurrence is highly variable

Processing of Thionin Precursors in Barley Leaves by a Vacuolar Proteinase

Thionins are synthesized as precursors with a signal peptide and a long C-terminal acidic peptide that is post-translationally processed. A fusion protein including the maltose-binding protein from Eschrrichia coli (MalE), thionin DG3 froin barley leaves, and its acidic C-terminal peptide has been used to obtain antibodies that recognize both domains of the precursor. In barley leaf sections. mature thionins accuinulated in the vacuolar content, while the acidic peptide was not detected in any cell fraction. Brefeldin A and inonensin inhibited processing of the precursor but its export from the microsomal fraction was not inhibited. Both purified vacuoles aiid an acid (pH 5.5) extract from leaves processed the fusion protein into a MalE-thionin and an acidic peptide fragment. A 70-kDa proteinase that effected this cleavage was purified froin the acid extract. Processing of the fusion protein by both lysed vacuoles and the purified proteinase was inhibited by Zn2+ and by Cu2+, but not by inhibitors of the previously described vacuolar processing thiol or aspartic proteinases. In vivo processing of the thionin precursor in leaf sections was also inhibited by Zn+, and Cu2+, Variants of the fusion protein with altered processing sites that represented thme of thionin precursors from different taxa were readily processed by the proteinase, whereas changing the polarity of either the C-terminal or N-terminal residues of the processing site prevented cleavage by the proteinase

Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples

A Naturally Occurring Plant Cysteine Protease Possesses Remarkable Toxicity against Insect Pests and Synergizes Bacillus thuringiensis Toxin

When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitating the movement of other insecticidal proteins in the diet to the midgut microvilli and thereby enhancing their toxicity. To directly determine the toxicity of Mir1-CP, the purified recombinant enzyme was directly tested against four economically significant Lepidopteran pests in bioassays. Mir1-CP LC50 values were 1.8, 3.6, 0.6, and 8.0 ppm for corn earworm, tobacco budworm, fall armyworm and southwestern corn borer, respectively. These values were the same order of magnitude as those determined for the Bacillus thuringiensis toxin Bt-CryIIA. In addition to being directly toxic to the larvae, 60 ppb Mir1-CP synergized sublethal concentrations of Bt-CryIIA in all four species. Permeabilization of the PM by Mir1-CP probably provides ready access to Bt-binding sites on the midgut microvilli and increases its activity. Consequently, Mir1-CP could be used for controlling caterpillar pests in maize using non-transgenic approaches and potentially could be used in other crops either singly or in combination with Bt-toxins

Immunological and Metabolomic Impacts of Administration of Cry1Ab Protein and MON 810 Maize in Mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab–induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab–specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight “cultivar” effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen

Transcriptome profiling of Pinus radiata juvenile wood with contrasting stiffness identifies putative candidate genes involved in microfibril orientation and cell wall mechanics

<p>Abstract</p> <p>Background</p> <p>The mechanical properties of wood are largely determined by the orientation of cellulose microfibrils in secondary cell walls. Several genes and their allelic variants have previously been found to affect microfibril angle (MFA) and wood stiffness; however, the molecular mechanisms controlling microfibril orientation and mechanical strength are largely uncharacterised. In the present study, cDNA microarrays were used to compare gene expression in developing xylem with contrasting stiffness and MFA in juvenile <it>Pinus radiata </it>trees in order to gain further insights into the molecular mechanisms underlying microfibril orientation and cell wall mechanics.</p> <p>Results</p> <p>Juvenile radiata pine trees with higher stiffness (HS) had lower MFA in the earlywood and latewood of each ring compared to low stiffness (LS) trees. Approximately 3.4 to 14.5% out of 3, 320 xylem unigenes on cDNA microarrays were differentially regulated in juvenile wood with contrasting stiffness and MFA. Greater variation in MFA and stiffness was observed in earlywood compared to latewood, suggesting earlywood contributes most to differences in stiffness; however, 3-4 times more genes were differentially regulated in latewood than in earlywood. A total of 108 xylem unigenes were differentially regulated in juvenile wood with HS and LS in at least two seasons, including 43 unigenes with unknown functions. Many genes involved in cytoskeleton development and secondary wall formation (cellulose and lignin biosynthesis) were preferentially transcribed in wood with HS and low MFA. In contrast, several genes involved in cell division and primary wall synthesis were more abundantly transcribed in LS wood with high MFA.</p> <p>Conclusions</p> <p>Microarray expression profiles in <it>Pinus radiata </it>juvenile wood with contrasting stiffness has shed more light on the transcriptional control of microfibril orientation and the mechanical properties of wood. The identified candidate genes provide an invaluable resource for further gene function and association genetics studies aimed at deepening our understanding of cell wall biomechanics with a view to improving the mechanical properties of wood.</p

Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed