HearNPV Pseudotyped with PIF1, 2, and 3 from MabrNPV: Infectivity and Complex Stability

Effective oral infection is set off by interaction of a group of conserved per os infectivity factors (PIFs) with larval midgut columnar epithelial cells. We constructed pseudotyped viruses by substituting pif1, pif2 or pif3 genes of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) with their homologs from Mamestra bracissae multiple nucleopolyhedrovirus and tested their infectivity to tissue culture cells and to larvae. Transfection and infection assays revealed that all recombinant viruses generated infectious budded virus in both cell culture and in larvae. Electron microscopy showed synthesized occlusion body and occlusion derived virus (ODV) were morphologically indistinguishable from those of the parental virus. By contrast, feeding assays revealed that pseudotyped viruses could not rescue oral infectivity except for pif3 pseudotyped virus that only partially rescued oral infectivity but at a mortality rate much lower than that of the parental HearNPV. Consistent with the bioassay result, PIF complex was detected in ODVs of pif3 pseudotyped virus only but not in pif1 or pif2 pseudotyped viruses. Our results suggest that PIF complex is essential for oral infectivity, and in the formation of the PIF complex, PIF1, 2 are virus-specific while PIF3 does not appear to be as specific and can function in heterologous environment, albeit to a much more limited extent.     

Expression of foreign gene by cysteine proteinase null recombinant baculovirus

The baculovirus expression vector systems (BEVS) are broadly used for producing foreign proteins in lepidopteran larvae. Most commercial BEVS are engineered to insert foreign genes into the polyhedrin (polh) locus and lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. Current knowledge in baculovirus genomics makes it possible to engineer BEVS into other parts of the virus genome. In our work, we have expressed recombinant M-HBsAg (middle surface antigen of human hepatitis B) in the baculovirus construct, rBmNPV-Deltav-cath-M-HBsAg, inserting foreign gene into the v-cath locus of the Bombyx mori nucleopolyhedrovirus (BmNPV) such that the v-cath gene is deleted and the native polh gene is retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced at a very late stage of infection.     

Molecular identification and phylogenetic analyses of multiple nucleopolyhedrovirus isolated from <Emphasis Type="Italic">Lymantria obfuscata</Emphasis> (Lepidoptera: Lymantriidae) in India

The Lymantria obfuscata Walker (Lyob) multiple (M) nucleopolyhedrovirus (NPV) (LyobMNPV) has been isolated and successfully applied for the management of the Indian gypsy moth, L. obfuscata in Jammu and Kashmir (J&K), India. The present work aimed to investigate the variability of LyobMNPV isolates from six localities of J&K through molecular [amplification of the polyhedrin (polh), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes] and biological (bioassays) characterization. To identify the position of LyobMNPV in the phylogenetic tree of baculoviruses, partial sequences of the polh, lef-8 and lef-9 genes were determined by using the DNA sequences within their coding regions by optimizing the polymerase chain reaction with degenerate primers. The sequence alignment revealed that LyobMNPV isolates exhibited seven, five and eleven single nucleotide polymorphic sites in the case of polh, lef-8 and lef-9, respectively. The phylogenetic analyses supported placing LyobMNPV with the Lymantria dispar L. MNPV (LdMNPV) isolates from different countries, and showed that it was more closely related to LdMNPV than to Lymantria xylina Swinhoe NPV and Lymantria monacha L. NPV. The contaminated diet plug bioassays using 2nd instar larvae indicated that the median lethal dose (LD₅₀) and median survival time (ST₅₀) of different isolates of LyobMNPV against L. obfuscata were lower than those of LdMNPV against L. dispar. LyobMNPV was more closely related to LdMNPV but its LD₅₀ and ST₅₀ were lower than those of LdMNPV. The study provides novel information on the position of LyobMNPV in the phylogenetic tree of baculoviruses and about biological and genetic variation of Lymantria species’ NPV isolates from different parts of the world.     

Alteration of a recombinant protein <Emphasis Type="Italic">N</Emphasis>-glycan structure in silkworms by partial suppression of <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase gene expression

Objective

To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae.

Results

Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man₃GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins.

Conclusions

Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Characterization of a <Emphasis Type="Italic">Pinus sylvestris</Emphasis> thaumatin-like protein gene and determination of antimicrobial activity of the in vitro expressed protein

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins, which are involved in plant defense responses to pathogen infection. Expression of the Pinus sylvestris L. TLP gene is up-regulated by methyl jasmonate treatment and inoculation with Heterobasidion annosum. A full-length Pinus taeda TLP gene sequence was used to design PCR primers for amplification of the full-length TLP gene from P. sylvestris. A putative 705-bp open reading frame of TLP gene was cloned into Escherichia coli cells, and then subcloned into the overexpression vector pET100 using BL21 Star expression bacteria. Optimization of the expression of recombinant TLP was achieved by decreasing both expression temperature and IPTG concentration. The purified 24.6-kDa TLP shows antimicrobial activity against 12 fungal species.     

Fine mapping <Emphasis Type="Italic">Ruv2</Emphasis>, a new rust resistance gene in cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>), to a 193-kb region enriched with NBS-type genes

Key message

A new rust resistance gene Ruv2 was fine-mapped in cowpea to a 193-kb region on chromosome 2, which harboured 23 predicted gene models enriched with NBS-type genes.

Abstract

ZN016 is a landrace vegetable cowpea highly resistant to rust. Two previous studies using mixed-spores inoculation suggested different modes of inheritance of rust resistance in ZN016. In this study, we initially developed a detached leaf assay with a purified single-rust isolate (Auv-LS). Using this approach, we assessed the inheritance of rust resistance in a recombinant inbred line (RIL) population and an F₂ population, both derived from the cross of “ZN016” and the susceptible cultivar “Zhijiang282.” A single dominant gene mode against Auv-LS was revealed in both populations. QTL mapping showed that this gene was coincident with the Ruv2 locus on LG7, one of the three resistance QTLs previously mapped based on mixed-spores inoculation data. Therefore, Ruv2 was considered as specifically against the rust isolate Auv-LS. Through an analysis of the RIL recombinants at Ruv2, we fine-mapped the gene to an ~?0.45-cM interval between SNP markers 2_09656 and 2_00973, which corresponded to an ~?193-kb region on chromosome 2 that harboured 23 predicted gene models enriched with NBS-type genes. Re-sequencing of the two parents revealed polymorphisms in four genes predictively to cause substantial protein structural changes, rendering them valuable candidate genes for future validation. Cross-species syntenic analysis indicated that Ruv2 may represent a novel rust resistance gene in food legumes. A cleaved amplified polymorphic sequences marker tightly linked to Ruv2 was developed to facilitate breeding. This work establishes a basis for map-based cloning of Ruv2 and breeding for rust resistance in cowpea and other legume crops.

     

Characterization of an Insecticidal Protein from <Emphasis Type="Italic">Withania somnifera</Emphasis> Against Lepidopteran and Hemipteran Pest

Lectins are carbohydrate-binding proteins with wide array of functions including plant defense against pathogens and insect pests. In the present study, a putative mannose-binding lectin (WsMBP1) of 1124 bp was isolated from leaves of Withania somnifera. The gene was expressed in E. coli, and the recombinant WsMBP1 with a predicted molecular weight of 31 kDa was tested for its insecticidal properties against Hyblaea puera (Lepidoptera: Hyblaeidae) and Probergrothius sanguinolens (Hemiptera: Pyrrhocoridae). Delay in growth and metamorphosis, decreased larval body mass and increased mortality was recorded in recombinant WsMBP1-fed larvae. Histological studies on the midgut of lectin-treated insects showed disrupted and diffused secretory cells surrounding the gut lumen in larvae of H. puera and P. sanguinolens, implicating its role in disruption of the digestive process and nutrient assimilation in the studied insect pests. The present study indicates that WsMBP1 can act as a potential gene resource in future transformation programs for incorporating insect pest tolerance in susceptible plant genotypes.     

Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous <Emphasis Type="Italic">GFP</Emphasis> genes in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura⁺ and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10^?8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.     

<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry11Ba works synergistically with Cry4Aa but not with Cry11Aa for toxicity against mosquito <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera: Culicidae) larvae

A 2,175-bp modified gene (cry11Ba-S1) encoding Cry11Ba from Bacillus thuringiensis subsp. jegathesan was designed and the recombinant protein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant Cry11Ba was highly toxic against Culex pipiens mosquito larvae, being nine and 17 times more toxic than mosquitocidal Cry4Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis, respectively. Interestingly, a further increase in the toxicity of the recombinant Cry11Ba was achieved by mixing with Cry4Aa, but not with Cry11Aa. These findings suggested that Cry11Ba worked synergistically with Cry4Aa, but not with Cry11Aa, in exhibiting toxicity against C. pipiens larvae. On the other hand, the amount of Cry toxin bound to brush border membrane vesicles (BBMVs) did not significantly change between individual toxins and the toxin mixtures, suggesting that the increase in toxins binding to BBMVs was not a reason for the observed synergistic effect. It is generally accepted that synergism of toxins is a potentially powerful tool for enhancing insecticidal activity and managing Cry toxin resistance in mosquitoes. The mixture of Cry4Aa and Cry11Ba in order to increase toxicity would be very valuable in terms of mosquito control.     

Genetic mapping and development of co-segregating markers of <Emphasis Type="Italic">RpsQ</Emphasis>, which provides resistance to <Emphasis Type="Italic">Phytophthora sojae</Emphasis> in soybean

Key message

The RpsQ Phytophthora resistance locus was finely mapped to a 118-kb region on soybean chromosome 3. A best candidate gene was predicted and three co-segregating gene markers were developed.

Abstract

Phytophthora root rot (PRR), caused by Phytophthora sojae, is a major threat to sustainable soybean production. The use of genetically resistant cultivars is considered the most effective way to control this disease. The Chinese soybean cultivar Qichadou 1 exhibited a broad spectrum resistance, with a distinct resistance phenotype, following inoculation with 36 Chinese P. sojae isolates. Genetic analyses indicated that the disease resistance in Qichadou 1 is controlled by a single dominant gene. This gene locus was designated as RpsQ and mapped to a 118-kb region between BARCSOYSSR_03_0165 and InDel281 on soybean chromosome 3, and co-segregated with Insert11, Insert144 and SNP276. Within this region, there was only one gene Glyma.03g27200 encoding a protein with a typical serine/threonine protein kinase structure, and the expression pattern analysis showed that this gene induced by P. sojae infection, which was suggested as a best candidate gene of RpsQ. Candidate gene specific marker Insert144 was used to distinguish RpsQ from the other known Rps genes on chromosome 3. Identical polymerase chain reaction amplification products were produced for cultivars Qichadou 1 (RpsQ) and Ludou 4 (Rps9). All other cultivars carrying Rps genes on chromosome 3 produced different PCR products, which all lacked a 144-bp fragment present in Qichadou 1 and Ludou 4. The phenotypes of the analyzed cultivars combined with the physical position of the PRR resistance locus, candidate gene analyses, and the candidate gene marker test revealed RpsQ and Rps9 are likely the same gene, and confer resistance to P. sojae.

     

<Emphasis Type="Italic">Pm61</Emphasis>: a recessive gene for resistance to powdery mildew in wheat landrace Xuxusanyuehuang identified by comparative genomics analysis

Key message

A single recessive powdery mildew resistance gene Pm61 from wheat landrace Xuxusanyuehuang was mapped within a 0.46-cM genetic interval spanning a 1.3-Mb interval of the genomic region of chromosome arm 4AL.

Abstract

Epidemics of powdery mildew incited by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt) have caused significant yield reductions in many wheat (Triticum aestivum)-producing regions. Identification of powdery mildew resistance genes is required for sustainable improvement of wheat for disease resistance. Chinese wheat landrace Xuxusanyuehuang was resistant to several Bgt isolates at the seedling stage. Genetic analysis based on the inoculation of Bgt isolate E09 on the F₁, F₂, and F_2:3 populations produced by crossing Xuxusanyuehuang to susceptible cultivar Mingxian 169 revealed that the resistance of Xuxusanyuehuang was controlled by a single recessive gene. Bulked segregant analysis and simple sequence repeat (SSR) mapping placed the gene on chromosome bin 4AL-4-0.80-1.00. Comparative genomics analysis was performed to detect the collinear genomic regions of Brachypodium distachyon, rice, sorghum, Aegilops tauschii, T. urartu, and T. turgidum ssp. dicoccoides. Based on the use of 454 contig sequences and the International Wheat Genome Sequence Consortium survey sequence of Chinese Spring wheat, four EST-SSR and seven SSR markers were linked to the gene. An F₅ recombinant inbred line population derived from Xuxusanyuehuang?×?Mingxian 169 cross was used to develop the genetic linkage map. The gene was localized in a 0.46-cM genetic interval between Xgwm160 and Xicsx79 corresponding to 1.3-Mb interval of the genomic region in wheat genome. This is a new locus for powdery mildew resistance on chromosome arm 4AL and is designated Pm61.

     

Construction of heterologous gene expression cassettes for the development of recombinant <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.     

Stability,oviposition attraction,and larvicidal activity of binary toxin from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus sphaericus produces a two-chain binary toxin composed of BinA (42 kDa) and BinB (51 kDa), which are deposited as parasporal crystals during sporulation. The toxin is highly active against Culex larvae and Aedes and Anopheles mosquitoes, which are the principal vectors for the transmission of malaria, yellow fever, encephalitis, and dengue. The use of B. sphaericus and Bacillus thuringiensis in mosquito control programs is limited by their sedimentation in still water. In this study, the binA and binB genes were cloned and the recombinant BinAB protein was expressed in three strains of Escherichia coli. These recombinant strains were used in a toxicity assay against Culex quinquefasciatus larvae. The highest expression level was achieved when both proteins were expressed in a single operon construct. The BinAB protein expressed in the E. coli Arctic strain showed higher larvicidal activity than either of the recombinant proteins from the E. coli Ril or pLysS strains. Furthermore, it had the highest oviposition attraction (49.1%, P?相似文献   

Association of variable <Emphasis Type="Italic">rs1801282</Emphasis> locus of <Emphasis Type="Italic">PPARG2</Emphasis> gene with diabetic nephropathy

The association of the variable rs1801282 locus of the PPARG2 gene (peroxisome proliferator-activated receptor gamma) with type 2 diabetes mellitus and its complications was analyzed in inhabitants of the Republic of Bashkortostan. The genotype frequencies of the variable rs1801282 locus of the PPARG2 gene did not significantly differ in groups of healthy persons and patients with type 2 diabetes in all three considered inheritance models (codominant, dominant, and recessive). At the same time, it was demonstrated that the risk of one of the diabetic complications, i.e., diabetic nephropathy, was associated with the variable rs1801282 locus of the PPARG2 gene. Diabetic nephropathy was more common in patients with the C/C genotype (62.7%) compared to the C/G and G/G genotypes (37.5%), P = 0.036. The G allele is protective in regard to diabetic nephropathy (OR = 0.36) in patients with type 2 diabetes mellitus.     

Development of near-isogenic lines with different alleles of Piz locus and analysis of their breeding effect under Yangdao 6 background

Rice blast is one of the most destructive diseases of rice. The most effective way of managing this disease is to develop resistant cultivars by introducing resistance genes into elite rice recipients. In this study, the near-isogenic lines (NILs) of six resistance alleles of the Piz locus (Pizt, Pi2, Pigm, Pi40, Pi9 and Piz) were constructed with Yangdao 6 as genetic background. Seedling inoculation tests showed that most of the NILs, namely NIL-Pi2, NIL-Pigm, NIL-Pi9, NIL-Pizt and NIL-Pi40, exhibited good resistance to blast with resistance frequencies (RFs) of over 82.50 %, execpt NIL-Piz which showed lower resistance with a RF of only 36.13 %. Furthermore, the improved-resistance NILs exhibited high similarity of their resistance spectra, with overlapping degrees of resistance spectrum (OD) of more than 75.83 %. However, the RF of panicle blast for all NILs decreased significantly compared with seedling blast in an artificial inoculation test. Although NIL-Pigm showed a higher panicle blast RF of 80 %, other NILs with outstanding performance in seedling blast resistance, namely NIL-Pizt, NIL-Pi2, NIL-Pi9 and NIL-Pi40, exhibited middle or low RFs of panicle blast with values from 56.67 to 33.30 %. Natural induction in a disease nursery showed a consistent trend in artificial inoculation results at seedling and heading stages. While NIL-Pigm was found to exhibit good resistance to leaf blast and panicle blast, NIL-Pi9 and NIL-Pizt were further demonstrated to show excellent resistance in Suichuan, Jiangxi province and Enshi, Hubei province, respectively, because of the race–region specificity. Agronomic traits of NILs were also investigated in order to evaluate the linkage drag effect of different alleles of the Piz locus. The resistance effects of the different alleles of the Piz locus under identical genetic background against seedling blast and panicle blast was first reported in this study, and the above results are expected to provide a theoretical support for the rational utilization of broad-spectrum resistance genes in breeding practice.