Amino acid substitution in α-helix 7 of Cry1Ac δ-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner

AbstractInsecticidal proteins or δ-endotoxins of Bacillus thuringiensis are highly toxic to a wide range of agronomically important pests. The toxins are formed of three structural domains. The N-terminal domain is a bundle of eight α-helices and is implicated in pore formation in insect midgut epithelial membranes. All the δ-endotoxins share a common hydrophobic motif of eight amino acids in α-helix 7. A similar motif is also present in fragment B of diphtheria toxin (DT). Site-directed mutagenesis of Cry1Ac δ-endotoxin of B. thuringiensis was carried out to substitute its hydrophobic motif with that of DT fragment B. The mutant toxin was shown to be more toxic to the larvae of Helicoverpa armigera (cotton bollworm) than the wild-type toxin. Voltage clamp analysis with planar lipid bilayers revealed that the mutant toxin opens larger ion channels and induces higher levels of conductance than the wild-type toxin

Transcriptomes of the Anther Sporophyte: Availability and Uses

An anther includes sporophytic tissues of three outer cell layers and an innermost layer, the tapetum, which encloses a locule where the gametophytic microspores mature to become pollen. The sporophytic tissues also comprise some vascular cells and specialized cells of the stomium aligning the long anther axis for anther dehiscence. Studies of the anther sporophytic cells, especially the tapetum, have recently expanded from the use of microscopy to molecular biology and transcriptomes. The available sequencing technologies, plus the use of laser microdissection and in silico subtraction, have produced high-quality anther sporophyte transcriptomes of rice, Arabidopsis and maize. These transcriptomes have been used for research discoveries and have potential for future discoveries in diverse areas, including developmental gene activity networking and changes in enzyme and metabolic domains, prediction of protein functions by quantity, secretion, antisense transcript regulation, small RNAs and promoters for generating male sterility. We anticipate that these studies with rice and other transcriptomes will expand to encompass other plants, whose genomes will be sequenced soon, with ever-advancing sequencing technologies. In comprehensive gene activity profiling of the anther sporophyte, studies involving transcriptomes will spearhead investigation of the downstream gene activity with proteomics and metabolomics

The AtXTH28 Gene, a Xyloglucan Endotransglucosylase/Hydrolase, is Involved in Automatic Self-Pollination in Arabidopsis thaliana

Successful automatic self-pollination in flowering plants is dependent on the correct development of reproductive organs. In the stamen, the appropriate growth of the filament, which largely depends on the mechanical properties of the cell wall, is required to position the anther correctly close to the stigma at the pollination stage. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of xyloglucan cross-links, thereby controlling the extensibility or mechanical properties of the cell wall in a wide variety of plant tissues. Our reverse genetic analysis has revealed that a loss-of-function mutation of an Arabidopsis XTH family gene, AtXTH28, led to a decrease in capability for self-pollination, probably due to inhibition of stamen filament growth. Our results also suggest that the role of AtXTH28 in the development of the stamen is not functionally redundant with its closest paralog, AtXTH27. Thus, our finding indicates that AtXTH28 is specifically involved in the growth of stamen filaments, and is required for successful automatic self-pollination in certain flowers in Arabidopsis thaliana

Gibberellin Acts through Jasmonate to Control the Expression of MYB21, MYB24, and MYB57 to Promote Stamen Filament Growth in Arabidopsis

Precise coordination between stamen and pistil development is essential to make a fertile flower. Mutations impairing stamen filament elongation, pollen maturation, or anther dehiscence will cause male sterility. Deficiency in plant hormone gibberellin (GA) causes male sterility due to accumulation of DELLA proteins, and GA triggers DELLA degradation to promote stamen development. Deficiency in plant hormone jasmonate (JA) also causes male sterility. However, little is known about the relationship between GA and JA in controlling stamen development. Here, we show that MYB21, MYB24, and MYB57 are GA-dependent stamen-enriched genes. Loss-of-function of two DELLAs RGA and RGL2 restores the expression of these three MYB genes together with restoration of stamen filament growth in GA-deficient plants. Genetic analysis showed that the myb21-t1 myb24-t1 myb57-t1 triple mutant confers a short stamen phenotype leading to male sterility. Further genetic and molecular studies demonstrate that GA suppresses DELLAs to mobilize the expression of the key JA biosynthesis gene DAD1, and this is consistent with the observation that the JA content in the young flower buds of the GA-deficient quadruple mutant ga1-3 gai-t6 rga-t2 rgl1-1 is much lower than that in the WT. We conclude that GA promotes JA biosynthesis to control the expression of MYB21, MYB24, and MYB57. Therefore, we have established a hierarchical relationship between GA and JA in that modulation of JA pathway by GA is one of the prerequisites for GA to regulate the normal stamen development in Arabidopsis

Analysis of the Plant bos1 Mutant Highlights Necrosis as an Efficient Defence Mechanism during D. dadantii/Arabidospis thaliana Interaction

Dickeya dadantii is a broad host range phytopathogenic bacterium provoking soft rot disease on many plants including Arabidopsis. We showed that, after D. dadantii infection, the expression of the Arabidopsis BOS1 gene was specifically induced by the production of the bacterial PelB/C pectinases able to degrade pectin. This prompted us to analyze the interaction between the bos1 mutant and D. dadantii. The phenotype of the infected bos1 mutant is complex. Indeed, maceration symptoms occurred more rapidly in the bos1 mutant than in the wild type parent but at a later stage of infection, a necrosis developed around the inoculation site that provoked a halt in the progression of the maceration. This necrosis became systemic and spread throughout the whole plant, a phenotype reminiscent of that observed in some lesion mimic mutants. In accordance with the progression of maceration symptoms, bacterial population began to grow more rapidly in the bos1 mutant than in the wild type plant but, when necrosis appeared in the bos1 mutant, a reduction in bacterial population was observed. From the plant side, this complex interaction between D. dadantii and its host includes an early plant defence response that comprises reactive oxygen species (ROS) production accompanied by the reinforcement of the plant cell wall by protein cross-linking. At later timepoints, another plant defence is raised by the death of the plant cells surrounding the inoculation site. This plant cell death appears to constitute an efficient defence mechanism induced by D. dadantii during Arabidopsis infection

A Regulatory Network for Coordinated Flower Maturation

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs

Isolation and analysis of genomic clones coding for legumin storage proteins and their copy number determination in chickpea, <i>Cicer arietinum </i>L.

1117-1122The genes encoding legumin storage proteins of chickpea were identified initially by Southern blot hybridization of restricted total genomic DNA of chickpea with pea legumin cDNA probes (pDUB6 and pDUB8). Since these probes showed strong homology to chickpea DNA, they were used for screening chickpea genomic library to isolate and ascertain the positive clones. Confirmation and detailed analysis of these clones were then done by restriction analysis and Southern hybridization. The gene copy number of legumin was estimated and found to be 10 to 12 in chickpea genome

Efficacy of lepidopteran specific δ-endotoxins of Bacillus thuringiensis against Helicoverpa armigera

This article does not have an abstract

A fusion gene coding for two different d-endotoxinsof Bacillus thuringiensis toxic to Plutella xylostella and usefulfor resistance management

Two truncated Bacillus thuringiensis δ-endotoxin genes, belonging to the classes cry1Ab and cry1B, and both coding for N-terminal toxic fragments of the corresponding crystal proteins, were translationally fused. Expression of the fusion gene driven by the cry1C promoter in Escherichia coli at a very high level resulted in a protein with enhanced toxicity to the diamondback moth (Plutella xylostella)

Bacillus thuringiensis cry1Ab gene confers resistance to potato against Helicoverpa armigera (Hubner)

Helicoverpa armigera is one of the important insect pests adversely affecting the yield of potatoes in India. A synthetic gene encoding the insecticidal crystal protein (Cry1Ab) of Bacillus thuringiensis (Bt) has been introduced into five genotypes of potato usingAgrobacterium tumefaciens. Southern analysis of DNA from transgenic plants confirmed the integration and copy number of the transgene. Double-antibody quantitative sandwich ELISA analysis demonstrated high levels of Cry1Ab protein expression in transgenic plants. Insect bioassays on the leaves of transgenic plants showed considerable protection against the larvae of H. armigera in terms of leaf area consumed and larval weight reduction