preparing-rearing-axenic-insects-with-tissue-cultured-seedlings-for

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

hubei university

Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an  cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the  transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5' untranslated region (UTR) from  of bacteriophage T7 and the transcript-stabilizing 3'UTR from the  ribosomal RNA operon . Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.

 Assembly in  of Transformation Vectors for Plastid Genome Engineering.

Plastid transformation technology has several attractive features compared with traditional nuclear transformation technology. However, only a handful of species are able to be successfully transformed. Here, we report an efficient and stable plastid transformation protocol for poplar, an economically important tree species grown worldwide. We transformed the Bacillus thuringiensis cry1C gene into the poplar plastid genome, and homoplasmic transplastomic poplar was obtained after two to three rounds of regeneration under antibiotic selection for 7-12 months. The transplastomic poplar expressing Cry1C insecticidal protein showed the highest accumulation level in young leaves, which reached up to 20.7 μg g-1 fresh weight, and comparatively low levels in mature and old leaves, and hardly detectable levels in non-green tissues, such as phloem, xylem and roots. Transplastomic poplar showed high toxicity to Hyphantria cunea and Lymantria dispar, two notorious forest pests worldwide, without affecting plant growth. These results are the first successful examples of insect-resistant poplar generation by plastid genome engineering and provide a new avenue for future genetic improvement of poplar plants.

Bacillus thuringiensis cry1C expression from the plastid genome of poplar leads to high mortality of leaf-eating caterpillars.

Insects' intestinal microbes have profound effects on the host's physiological traits, which can impact their physiology at both the local (gut) and systemic (body) levels. Nevertheless, the molecular mechanisms underlying host-microbiota interactions, especially in non-model insects, remain elusive. Recently, tissue-specific transcriptomic analysis has been highlighted as a robust tool in studying host-microbe interactions. Plagiodera versicolora is a worldwide leaf-eating pest that primarily feeds on willows and poplar. The interplay between gut microflora and this host beetle has yet to be studied. Herein, we investigate the effects of the gut microbiota on the body mass of P. versicolora larvae, compare the nutrition status of larvae in absence and presence of gut microbiota, and profile gut bacterial loads throughout its developmental larval stages. We then perform comparative transcriptomic analysis of gut and body tissues in axenic and non-axenic larvae. Finally, we confirm the expression patterns of representative genes in nutritional metabolism and immunity. Results show that weight growth is retarded in conventional larvae, with a concomitant increase of total bacterial load by the 5 development day, and germ-free larvae have a higher glucose content than conventional-reared larvae. Both nutritional and immunological analyses indicate that gut bacteria are a burden in the beetle's larval development. These findings elucidate the impacts of gut microbiota on P. versicolora, and provide insight into tissue-specific responses to gut microflora in this pest at the genetic level, boosting our understanding of the molecular mechanisms underlying host-microbe interactions in leaf beetles and beyond.

Metabolic and immunological effects of gut microbiota in leaf beetles at the local and systemic levels.

The wide-spread culture of transgenic Bt cotton resisting the infamous cotton bollworms has reduced the adoption of broad-spectrum insecticides to a large extent. Consequently, the non-targeted insect  Jakovlev has become a major cotton pest in China. Entomopathogenic microbes show promising results for controlling this pest in the future, but  innate immune responses to these pathogens are poorly understood. Here, we used the entomopathogenic fungus  and the Gram-negative pathogenic bacteria  to infect  nymphs, followed by high throughput RNA-seq to analyze the immune transcriptomes of  in response to the two pathogens. A total of 150 immunity-related genes were identified, including pattern recognition receptors, extracellular signal modulators, signal pathways (Toll, IMD, JNK, and JAK/STAT), and response effectors. Further quantitative real-time PCR analysis demonstrated that  and  were recognized by different receptors (GNBP and PGRP, respectively); activated Toll pathway and IMD pathway respectively; and both induced expression of the effector gene Defensin. However, melanization is suppressed in -infected nymphs. Collectively, this study provides a transcriptomic snapshot of the  immune system, and at the genetic level, gains multifaceted insights of the immune response to fungal and Gram-negative bacterial pathogens. Ultimately this work pioneers the study of molecular mechanisms underlying immune interactions between  and its pathogens and assists in the development of novel mitigation strategies to control this pest.

Article	Year
Assembly in of Transformation Vectors for Plastid Genome Engineering. Frontiers in plant science\| PubMed ID: 28871270	2017
Bacillus thuringiensis cry1C expression from the plastid genome of poplar leads to high mortality of leaf-eating caterpillars. Tree physiology\| PubMed ID: 31222266	2019
Metabolic and immunological effects of gut microbiota in leaf beetles at the local and systemic levels. Integrative zoology\| PubMed ID: 33704889	2021
Comparative Analysis of Jakovlev (Hemiptera: Miridae) Immune Responses to Fungal and Bacterial Pathogens. Frontiers in physiology\| PubMed ID: 33815150	2021

Meiqi Ma

Biography

JoVE Journal Publications

Other Publications