Yueqing Cao

Role of hunchback in segment patterning of Locusta migratoria manilensis revealed by parental RNAi

In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, we have investigated the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (i) L. migratoria manilensis hb (Lmm’hb) controls germ band morphogenesis and segmentation in the anterior region; (ii) Lmm’hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (iii) Lmm’hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila.

Cloning of a novel protease required for the molting of Locusta migratoria manilensis

Molting is required for progression between larval stages in the life cycle of an insect. The essence of insect molting is the laying down of new cuticle followed by shedding of the old cuticle. Degradation and recycling of old cuticle are brought about by enzymes present in the molting fluid, which fills the space between the old and new cuticle. Here, we describe the cloning of a novel protease gene from Locusta migratoria manilensis, designated as Lm‐TSP. The cDNA and its deduced protein sequences were deposited in GenBank (accession numbers EF081255 and ABN13876, respectively). Sequence analysis indicated that Lm‐TSP belongs to the trypsin‐like serine protease family. We show, by RNA interference (RNAi), that silencing of Lm‐TSP leads to dramatic reductions in protease and cuticle‐degrading activity of a molting fluid, which leads to molting defects from fourth‐instar larvae (L4) to fifth‐instar larvae (L5), and between L5 and adult stages. These observations suggest that Lm‐TSP plays a critical role in L. migratoria manilensis ecdysis.

Genetically altering the expression of neutral trehalase gene affects conidiospore thermotolerance of the entomopathogenic fungus Metarhizium acridum

BackgroundThe entomopathogenic fungus Metarhizium acridum has been used as an important biocontrol agent instead of insecticides for controlling crop pests throughout the world. However, its virulence varies with environmental factors, especially temperature. Neutral trehalase (Ntl) hydrolyzes trehalose, which plays a role in environmental stress response in many organisms, including M. acridum. Demonstration of a relationship between Ntl and thermotolerance or virulence may offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi through genetic engineering.ResultsWe selected four Ntl over-expression and four Ntl RNA interference (RNAi) transformations in which Ntl expression is different. Compared to the wild-type, Ntl mRNA expression was reduced to 35-66% in the RNAi mutants and increased by 2.5-3.5-fold in the over-expression mutants. The RNAi conidiospores exhibited less trehalase activity, accumulated more trehalose, and were much more tolerant of heat stress than the wild-type. The opposite effects were found in conidiospores of over-expression mutants compared to RNAi mutants. Furthermore, virulence was not altered in the two types of mutants compared to the wild type.ConclusionsNtl controlled trehalose accumulation in M. acridum by degrading trehalose, and thus affected conidiospore thermotolerance. These results offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi without affecting virulence.

Analysis of the Intestinal Microflora in Hepialus gonggaensis Larvae Using 16S rRNA Sequences

Gut microbial diversity provides insight into the basic function of a gut microbial ecosystem. In this study, restriction fragment length polymorphism 16S rRNA sequences was used to detect the intestinal microbial diversity of Hepialus gonggaensis larvae. The total DNA of microorganisms was extracted from the intestinal contents and 16S rRNA was amplified. A nearly full-length of 16S rRNA sequence library was constructed. The fingerprints of the microorganisms were analyzed by isolating plasmid and then digesting them with EcoRI, MspI, and HaeIII enzymes, respectively. The library established includes 35 restriction endonuclease types and a phylogenetic tree depicted the linkage of the isolated microbial from the guts of H. gonggaensis larvae. The dominant bacteria in the guts of H. gonggaensis larvae belong to Rahnella sp and Carnobacterium sp and accounted for 45.58% and 30.88% of the total 16S rRNA clones library, respectively. The result showed that bacteria diversity in the guts of H. gonggaensis larvae had some differences from those isolated from normal environment.

Mmc, a gene involved in microcycle conidiation of the entomopathogenic fungus Metarhizium anisopliae.

Microcycle conidiation is a survival mechanism for some fungi encountering unfavorable conditions, in which asexual spores germinate secondary spores directly without formation of mycelium. Here, we isolated a microcycle conidiation associated gene, Mmc, from Metarhizium anisopliae and obtained its full length of cDNA and DNA sequence. To clarify its roles in conidiation, we constructed an Mmc RNA interference (RNAi) vector with dual promoter system to knockdown Mmc transcript level, and then analyzed RNAi mutant phenotypes. On microcycle conidiation medium, the RNAi mutant performed normal conidiation instead of microcycle conidiation with significantly reduced growth speed and conidia yield of 5.29-fold and 3.18-fold lower, respectively, than that of the wild-type. Meanwhile, on normal conidiation medium, no significant difference was found in conidiation speed and total yield between the wild-type and RNAi mutant. These data demonstrated that the Mmc gene regulated microcycle conidiation but did not affect normal conidiation. In addition, results of heat treatment, UV-B radiation and bioassays of RNAi mutant indicated that Mmc was also involved in heat resistance but irrelevant to UV-B tolerance and virulence of M. anisopliae. This study helped understanding the regulation of sporulation of the entomopathogenic fungus M. anisopliae.

The tetraspanin gene MaPls1 contributes to virulence by affecting germination, appressorial function and enzymes for cuticle degradation in the entomopathogenic fungus, Metarhizium acridum

In most eukaryotes, tetraspanins regulate cellular activities by associating with other membrane components. In phytopathogenic fungi, the tetraspanin Pls1 controls appressorium-mediated penetration. However, regulation of Pls1 and its associated signalling pathways are not clear. In this study, the MaPls1 gene from the entomopathogenic fungus Metarhizium acridum was functionally characterized. MaPls1 was highly expressed in mycelium and appressorium, and accumulated on the plasma membrane or in the cytoplasm. Compared with a wild-type strain, the deletion mutant ΔMaPls1 had delayed germination and appressorium formation and impaired turgor pressure on locust wings, but normal germination on medium and non-host insect matrices. Bioassays showed that ΔMaPls1 had decreased virulence and hyphal body formation in haemolymph when topically inoculated, but was not different from wild type when the insect cuticle was bypassed. Moreover, the ability to grow out of the cuticle was impaired in ΔMaPls1. Digital gene expression profiling revealed that genes involved in hydrolysing host cuticle and cell wall synthesis and remodelling were downregulated in ΔMaPls1. MaPls1 participated in crosstalk with signalling pathways such as the cyclic adenosine monophosphate-dependent protein kinase A and calmodulin-dependent pathways. Taken together, these results demonstrated the important roles of MaPls1 at the early stage of infection-associated development in M. acridum.

Trehalose-6-phosphate synthase 1 from Metarhizium anisopliae: clone, expression and properties of the recombinant

Trehalose, an important component in fungal spores, is a disaccharide which protects against several environmental stresses, such as heat, desiccation, salt. Trehalose-6-phosphate synthase 1 (TPS1) is a subunit of trehalose synthase complex in fungi; it plays a key role in the biosynthesis of trehalose. In this study, a full-length cDNA from Metarhizium anisopliae encoding TPS1 (designated as MaTPS1) was isolated. The MaTPS1 cDNA is composed of 1836 nucleotides encoding a protein of 517 amino acids with a molecular mass of 58 kDa. The amino acid sequence has a relatively high homology with the TPS1s in several other filamentous fungi. Southern blot analysis showed that MaTPS1 gene occurs as a single copy in the M. anisopliae genome. And MaTPS1 was cloned into Pichia pastoris KM71 and secretively expressed with a histamine tag to facilitate a rapid purification of recombinant MaTPS1 (designated reTPS1). The properties of reTPS1 were examined. The K(m) value of reTPS1 for UDP-glucose and glucose-6-phosphate was 9.6 mM and 3.9 mM, respectively, and the optimal pH and temperature were about 6.5 and 40 degrees C. The enzyme was highly specific to glucose-6-phosphate for glucosyl acceptor, and its activity decreased rapidly as the concentrations of phosphate increased. The expression system will provide sufficient amounts of reTPS1 for future structural characterization of the protein and use for further investigation of MaTPS1s function.

Optimization of parental RNAi conditions for hunchback gene in Locusta migratoria manilensis (Meyen)

Abstract  An investigation on the optimization of parental RNA interference (RNAi) conditions for hunchback (hb) gene in Locusta migratoria manilensis (Meyen) was conducted. Double stranded RNA (dsRNA) corresponding to hb gene was injected into haemocoel of female adults of L. migratoria manilensis. Embryos developed from the eggs laid by the injected adults on the 7th day after eclosion showed observable effects of RNAi for hb. The silencing effect after delivery treatment of dsRNA for hb gene was maintained for more than 21 days. A significant decrease of hb transcripts was further confirmed by Northern blot analysis. The dose of dsRNA/insect at 2 μg could trigger 96.9% RNAi effects, while silencing appeared to have no dependence on the size of dsRNA. Results suggest that parental RNAi could be employed to efficiently identify the developmental gene functions in L. migratoria manilensis.

Transformation of Metarhizium anisopliae with benomyl resistance and green fluorescent protein genes provides a tag for genetically engineered strains

A plasmid, pBGFP, carrying green fluorescent protein (gfp) and benomyl-resistance genes was constructed and transformed into Metarhizium anisopliae. The transformants grew normally and GFP fluorescence was detected. No change was found in virulence for the transformants. Fluorescence was detected in hyphae from the haemolymph of the infected locust, and the benomyl-resistance was maintained. Results suggested that the two markers provided a useful tool for screening and monitoring the engineered strains even after infection.

Calcineurin modulates growth, stress tolerance, and virulence in Metarhizium acridum and its regulatory network

Calcineurin is highly conserved and regulates growth, conidiation, stress response, and pathogenicity in fungi. However, the functions of calcineurin and its regulatory network in entomopathogenic fungi are not clear. In this study, calcineurin was functionally analyzed by deleting the catalytic subunit MaCnA from the entomopathogenic fungus Metarhizium acridum. The ΔMaCnA mutant had aberrant, compact colonies and blunt, shortened hyphae. Conidia production was reduced, and phialide differentiation into conidiogenous cells was impaired in the ΔMaCnA mutant. ΔMaCnA had thinner cell walls and greatly reduced chitin and β-1,3-glucan content compared to the wild type. The ΔMaCnA mutant was more tolerant to cell wall-perturbing agents and elevated or decreased exogenous calcium but less tolerant to heat, ultraviolet irradiation, and caspofungin than the wild type. Bioassays showed that ΔMaCnA had decreased virulence. Digital gene expression profiling revealed that genes involved in cell wall construction, conidiation, stress tolerance, cell cycle control, and calcium transport were downregulated in ΔMaCnA. Calcineurin affected some components of small G proteins, mitogen-activated protein kinase, and cyclic AMP (cAMP)-protein kinase A signaling pathways in M. acridum. In conclusion, our results gave a global survey of the genes downstream of calcineurin in M. acridum, providing molecular explanations for the changes in phenotypes observed when calcineurin was deleted.