Crystal structure and mode of action of a class V chitinase from <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

A class V chitinase from Nicotiana tabacum (NtChiV) with amino acid sequence similar to that of Serratia marcescens chitinase B (SmChiB) was expressed in E. coli and purified to homogeneity. When N-acetylglucosamine oligosaccharides [(NAG)_n] were hydrolyzed by the purified NtChiV, the second glycosidic linkage from the non-reducing end was predominantly hydrolyzed in a manner similar to that of SmChiB. NtChiV was shown to hydrolyze partially N-acetylated chitosan non-processively, whereas SmChiB hydrolyzes the same substrate processively. The crystal structure of NtChiV was determined by the single-wavelength anomalous dispersion method at 1.2 Å resolution. The protein adopts a classical (β/α)₈-barrel fold (residues 1–233 and 303–348) with an insertion of a small (α + β) domain (residues 234–302). This is the first crystal structure of a plant class V chitinase. The crystal structure of the inactive mutant NtChiV E115Q complexed with (NAG)₄ was also solved and exhibited a linear conformation of the bound oligosaccharide occupying ?2, +1, +2, and +3 subsites. The complex structure corresponds to an initial state of (NAG)₄ binding, which is proposed to be converted into a bent conformation through sliding of the +1, +2, and +3 sugar units to ?1, +1, and +2 subsites. Although NtChiV is similar to SmChiB, the chitin-binding domain is present in the C-terminus of the latter, but not in the former. Aromatic amino acid residues found in the substrate binding cleft of SmChiB, including Trp97, are substituted with aliphatic residues in NtChiV. These structural differences appear to be responsible for NtChiV being a non-processive enzyme.     

Interactions of Algae within the Community of <Emphasis Type="Italic">Gracilaria gracilis</Emphasis> (Rhodophyta)

Interactions among the unattached red alga Gracilaria gracilis, the dominant species of an algal community, and associated algal species Chaetomorpha linum, Enteromorpha prolifera f. prolifera, and Polysiphonia sp. were studied during and after an algal bloom. It was shown that during their bloom the associated algae Enteromorpha and Polysiphonia sp. significantly decreased the photosynthetic rate of G. gracilis but did not affect its growth rate. It is suggested that the inhibition of Gracilaria gracilis photosynthesis is connected to the impact of extracellular metabolites excreted by Chaetomorpha linum, Enteromorpha prolifera f. prolifera, and Polysiphonia sp. In laboratory experiments, the photosynthetic rate of the associated species was enhanced in the presence of Gracilaria. However, no significant alterations were observed in the content of chlorophyll a, growth, and the dark respiration rates of associated algae when they were kept together with Gracilaria. It was suggested that allelopathic interactions observed among algal species during the formation of the monospecific Gracilaria community, as well as during algal blooms, are not determinative.     

Purification and partial characterization of intact and truncated chitinase from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> HZP7 expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To ascertain the effect of chitin-binding domain (ChBD) and fibronectin type III domain (FN3) on the characterization of the intact chitinase from Bacillus thuringiensis.

Results

An intact chitinase gene (chi74) from B. thuringiensis HZP7 and its truncated genes (chi54, chi63 and chi66) were expressed in Escherichia coli BL21. The expression products were analyzed after purification. All chitinases were active from pH 4–7.5 and from 20 to 80 °C with identical optimal: pH 5.5 and 60 °C. The activity of colloid chitin degradation for Chi74 was the highest, followed by Chi66, Chi63 and Chi54. Ag⁺ reduced the activity of Chi74, Chi54, Chi63 and Chi66, but Mg²⁺ enhanced them. The effect of Ag⁺ and Mg²⁺ was more significant on the activity of Chi54 than on the activities of Chi63, Chi66 and Chi74.

Conclusion

ChBD_Chi74 and FN3_Chi74 domains play a role in exerting enzymatic activity and can improve the stability of chitinase.

     

Co-transformation of canola by chimeric chitinase and <Emphasis Type="Italic">tlp</Emphasis> genes towards improving resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T₂ generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.     

Revision of <Emphasis Type="Italic">Podocotyloides</Emphasis> Yamaguti, 1934 (Digenea: Opecoelidae), resurrection of <Emphasis Type="Italic">Pedunculacetabulum</Emphasis> Yamaguti, 1934 and the naming of a cryptic opecoelid species

Despite morphological and ecological inconsistencies among species, all plagioporine opecoelids with a pedunculate ventral sucker are currently considered to belong in the genus Podocotyloides Yamaguti, 1934. We revise the genus based on combined morphological and phylogenetic analyses of novel material collected from haemulid fishes in Queensland waters that we interpret to represent species congeneric with the type-species, Pod. petalophallus Yamaguti, 1934, also known from a haemulid, off Japan. Our phylogenetic analysis demonstrates polyphyly of Podocotyloides; prompts us to resurrect Pedunculacetabulum Yamaguti, 1934; and suggests that Pod. brevis Andres & Overstreet, 2013, from a deep-sea congrid in the Caribbean, and Pod. parupenei (Manter, 1963) Pritchard, 1966 and Pod. stenometra Pritchard, 1966, from mullids and chaetodontids, respectively, on the Great Barrier Reef, may each represent a distinct genus awaiting recognition. Our revised concept of Podocotyloides requires a pedunculate ventral sucker, but also a uterine sphincter prior to the genital atrium, a petalloid cirrus appendage, restriction of the vitelline follicles to the hindbody, and for the excretory vesicle to reach to the level of the ventral sucker. Of about 20 nominal species, we recognise just three in Podocotyloides (sensu stricto): Pod. petalophallus, Pod. gracilis (Yamaguti, 1952) Pritchard, 1966 and Pod. magnatestes Aleshkina & Gaevskaya, 1985. We provide new records for Pod. gracilis, and propose two new species of Podocotyloides, Pod. australis n. sp. and Pod. brevivesiculatus n. sp., and one new Pedunculacetabulum species, Ped. inopinipugnus n. sp., all from haemulids. Podocotyloides australis is morphologically indistinguishable from Pod. gracilis, and exploits the same definitive host, but is genetically and biogeographically distinct. It is thus a cryptic species, the first such opecoelid to be formally named.     

Resistance of transgenic zoysiagrass overexpressing the zoysiagrass class II chitinase gene <Emphasis Type="Italic">Zjchi2</Emphasis> against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG2-2 (IV)

Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass species used in golf courses and athletic fields. However, zoysiagrass is susceptible to large patch disease caused by Rhizoctonia solani AG2-2 (IV). Chitinases are pathogen-related (PR) proteins induced by viruses, bacteria, and fungi that hydrolyze chitin. Recently, we isolated a class II chitinase gene (Zjchi2) from zoysiagrass. The purified recombinant Zjchi2 showed broad-spectrum activity against various fungi, including R. solani AG2-2 (IV). In the current study, we generated transgenic zoysiagrass overexpressing Zjchi2 and then verified the resistance of transgenic plants to R. solani AG2-2 (IV). Polymerase chain reaction and Southern blot hybridization showed the integration of transgenes in zoysiagrass genomes and constitutive expression of Zjchi2, respectively. Antifungal activity was enhanced significantly in the transgenic zoysiagrass compared with wild-type plants. To our knowledge, this report is the first on the antifungal activity of a class II chitinase in transgenic zoysiagrass.     

A comparative study of transgenic canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) harboring either chimeric or native <Emphasis Type="Italic">Chit</Emphasis>42 genes against phytopathogenic fungi

Canola (Brassica napus L.), an agro-economically important crop in the world, is sensitive to many fungal pathogens. One strategy to combat fungal diseases is genetic engineering through transferring genes encoding the pathogenesis-related (PR) proteins such as chitinase which cause the chitin degradation of fungal cell wall. Chitinase Chit42 from Trichoderma atroviride (PTCC5220) plays an important role in biocontrol and has high antifungal activity against a wide range of phytopathogenic fungi. This enzyme lacks a chitin binding domain (ChBD) which is involved in binding activity to insoluble chitin. In the present study, we investigated the effect of chitin binding domain fused to Chit42 when compared with native Chit42. These genes were over-expressed under the CaMV35S promoter in B. napus, R line Hyola 308. Transformation of cotyledonary petioles was achieved by pBISM2 and pBIKE1 constructs containing chimeric and native Chit42 genes respectively, via Agrobacterium method. The insertion of transgenes in T₀ generation was verified through polymerase chain reaction (PCR) and Southern blot analysis. Antifungal activity of expressed chitinase in transgenic plants was also investigated by bioassays. The transgenic canola expressing chimeric chitinase showed stronger inhibition against phytopathogenic fungi that indicates the role of chitin binding domain.     

Molecular cloning and characterization of chitinase genes from zoysiagrass (<Emphasis Type="Italic">Zoysia Japonica</Emphasis> Steud.)

Zoysiagrass (Zoysia Japonica Steud.) is used frequently in golf courses and athletic fields. However, Zoysiagrass suffers from large-patch disease caused by Rhizoctonia solani AG2-2 (IV), which results in physical and economic loss. In this study, two full-length chitinase genes encoding pathogen-related proteins were isolated from zoysiagrass. Structural and expression analyses of these genes were carried out. The two isolated chitinases were classified into class Ib (Zjchi1) and class II (Zjchi2). Zjchi1 and, Zjchi2 expression was high in root and stolen and was induced in seedlings by Rhizoctonia solnai AG2-2 (IV) infection. To assess their antifungal activity, the two chitinases were overexpressed in Escherichia coli and purified using Ni²⁺ and glutathione affinity column chromatography. The purified recombinant chitinases showed broad-spectrum antifungal activity against Rhizoctonia solnai AG2-2 (IV), Rhizoctonia solnai AG-1 (IA), Rhizoctonia cerealis, Botrytis cinerea, Fusarium culmorum, Fusarium graminearum and Trichoderma reesei.     

Evaluation of antifungal,phosphate solubilisation,and siderophore and chitinase release activities of endophytic fungi from <Emphasis Type="Italic">Pistacia vera</Emphasis>

Fungal endophytes use different strategies to protect host plants from abiotic and biotic stress. In this study, we isolated endophytic fungi from Pistacia vera and characterised their antifungal activity against Aspergillus flavus, Rhizoctonia solani and Sclerotinia sclerotiorum, and their release of some factors that can alter plant growth capability. Trichoderma harzianum TH 5-1-2, T. harzianum TH 10-2-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition percentages in dual culture assays against A. flavus, R. solani and S. sclerotiorum, respectively. Among the fungal endophyte cultures, ethyl acetate extracts of T. harzianum TH 10-2-2, T. harzianum TH 5-1-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition of S. sclerotiorum, R. solani and A. flavus, respectively. Phosphate solubilisation was induced by Byssochlamys nivea BN 1-1-1 in culture. Large amounts of siderophore production were observed with Quambalaria cyanescens QC 11-3-2 and Epicoccum nigrum EN1, but Trichoderma spp. also produced siderophore in lower amounts. Trichoderma harzianum TH 5-1-2 produced the highest chitinase activity (2.92 U/mL). In general, among the endophytes isolated, Trichoderma spp. appear to have the most promise for promoting healthy growth of P. vera.     

Expression analysis on mycoparasitism related genes during antagonism of <Emphasis Type="Italic">Trichoderma</Emphasis> with <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis> causing red rot in sugarcane

Present study was aimed to select a suitable Trichoderma isolate as candidate antagonist based on its efficacy in producing cell wall degrading enzymes (CWDEs), its mycoparasitism activity and expression of related genes against the red rot pathogen caused by Colletotrichum falcatum in sugarcane. For which, six different isolates of Trichoderma selected from our earlier studies (T. harzianum, T. asperullum) were evaluated based on their capability in releasing cell wall degrading enzymes individually and during antagonism with C. falcatum in dual plate. Amongst T. harzianum (T20) exhibited the greatest mycoparasitic potential against the C. falcatum, by producing higher concentration of  CWDEs viz., chitinase and β-1, 3-glucanase, slightly lower amounts of cellulase and protease with significant reduction in polygalacturonase produced by pathogen. Further microscopic observation on interaction of C. falcatum with the selected isolate of T. harzianum (T20) exhibited the mycoparasitic activity of antagonist over pathogen in dual culture and inhibition of C. falcatum pathogenesis in detached sugarcane leaves. In addition, expression pattern of eight genes coding various enzymes involved in mycoparasitism by T. harzianum over C. falcatum were analyzed using qRT-PCR in vitro and on sugarcane leaves. In in vitro interactions, five genes of  cell wall degrading enzymes viz., chitinase (chit33), endochitinase (endo42), β-1, 3-glucanase (glu), exochitinase 1 (exc1), exochitinase 2 (exc2), were upregulated during and after contact as compared to before contact, while three genes related with proteases such as alkaline proteinase (prb1), trypsin-like protease (Pra1), subtilin-like serine protease (ssp), genes were upregulated during the contact with C. falcatum and slightly down regulated after contact. In detached leaves, seven genes were potentially upregulated except subtilin-like serine protease, which was down regulated during interaction of C. falcatum and T. harzianum as compared to T. harzianum inoculation alone. All these biochemical and molecular results confirm the efficacy of T. harzianum (T20) against C. falcatum and justify the right selection of candidate antagonist for our further studies on identification of antifungal genes/proteins against C. falcatum in sugarcane.     

Revision of Jaegeria (Compositae-Heliantheae)

The genusJaegeria consists of a diploid-polyploid complex of eight species found in moist habitats from Mexico to South America. The distributions, habitats, generic affinities, and tribal placement are discussed. Chromosome numbers were determined for all species exceptJ. axillaris, which is known only from Colombia.J. glabra andJ. pedunculata are diploid (2n = 18) ;J. hirta, J. macrocephala, J. gracilis, andJ. crassa are tetraploid (2n = 36) ;J. bellidiflora is octoploid (2n = 72). One Galápagos Island endemic,J. gracilis, is quite similar to the common mainland species,J. hirta, but the other endemic,J. crassa, is quite distinct from any other species.J. bellidijlora is most similar morphologically toJ. glabra;J. macrocephala is allied toJ. pedunculata. J. axillaris is somewhat distinct from the other species.     

Opisthobranch gastropods from the Lower Cretaceous of the Ulyanovsk Volga Region: 1. Genus <Emphasis Type="Italic">Tornatellaea</Emphasis> Conrad

Five new species of the genus Tornatellaea from the Lower Cretaceous of the Volga Region near Ulyanovsk, T. kabanovi sp. nov. (Hauterivian), T. densistriata sp. nov., T. gracilis sp. nov., T. volgensis sp. nov. (Barremian), and T. sinzovi sp. nov. (Aptian), are described. The data on geographical and stratigraphical distribution of new taxa are provided.     

Pitcher plant facilitates prey capture in a sympatric congener

Carnivorous plants avoid below-ground competition for nitrogen by utilizing an alternative nitrogen resource—invertebrate prey, but it remains unclear if sympatric carnivorous plants compete for prey resources. The aim of this study was to investigate if exploitative prey-resource competition occurs between the two sympatric pitcher plant species, Nepenthes rafflesiana and N. gracilis in Singapore. We first investigated if prey-resource partitioning occurs between these two species, and then investigated niche shift in N. gracilis by examining its pitcher contents along an in situ gradient of N. rafflesiana interspecific competition. Our results showed clear evidence of resource partitioning between the two species, but contrary to the expectation of competition, proximity to N. rafflesiana pitchers correlated with higher total prey numbers in N. gracilis pitchers. Our multivariate model of prey assemblages further suggested that N. rafflesiana facilitates N. gracilis prey capture, especially in several ant taxa that are trapped by both species. Concurrently, we found strong evidence for intraspecific competition between N. gracilis pitchers, suggesting that prey resources are exhaustible by pitcher-predation. Our results show that resource partitioning can be associated with facilitative interactions, instead of competition as is usually assumed. Facilitation is more typically expected between phylogenetically distant species, but divergences in resource acquisition strategies can permit facilitation between congeners.     

Acaricidal property of the essential oil from <Emphasis Type="Italic">Lippia gracilis</Emphasis> against <Emphasis Type="Italic">Tetranychus urticae</Emphasis> and a natural enemy, <Emphasis Type="Italic">Neoseiulus californicus</Emphasis>, under greenhouse conditions

The essential oil from the leaves of Lippia gracilis was investigated for fumigant and residual activity against Tetranychus urticae (Acari: Tetranychidae) and Neoseiulus californicus (Acari: Phytoseiidae). The results were compared to eugenol, Ortus^® and Azamax^®, as positive controls. Gas chromatography (GC) and GC/mass spectrometry analysis enabled the identification of 28 compounds, accounting for 99.1?±?0.6% of the essential oil. The major constituents were carvacrol (61%), p-cymene (11%) and thymol (11%). Mites were more susceptible to the oil in fumigant tests than in residual tests. Among the components, thymol and β-caryophyllene had the greatest fumigant and residual toxicity against T. urticae, respectively. The role of selected constituents (carvacrol, p-cymene, thymol, limonene, β-pinene, 1,8-cineole, terpinolene and β-caryophyllene) in the acaricidal properties of the L. gracilis essential oil is also discussed. Fumigant and residual effects of Lippia oil were more selective than eugenol with regard to a natural enemy of T. urticae, Neoseiulus californicus. Experiments under greenhouse conditions demonstrated greater toxicity of the Lippia oil in comparison to the positive control at 24, 48 and 72 h after treatment. The results suggest that Lippia oil is a good candidate for the formulation of a botanical acaricide for the integrated management of T. urticae.