Can intra‐specific genetic variation in arbuscular mycorrhizal fungi (Glomus etunicatum) affect a mesophyll‐feeding herbivore (Tupiocoris notatus Distant)?

Abstract.  1. Arbuscular mycorrhizal fungal (AMF) infection can have negative, positive or neutral effects on insect herbivore populations, but patterns are difficult to predict.
2. Intra-specific genetic variation in nutrient uptake ability between fungal isolates may also have indirect effects on insect herbivores due to changes in plant quality. In preliminary studies mirid ( Tupiocoris notatus ) populations were significantly reduced on tobacco ( Nicotiana rustica ) colonised by AMF but it was unknown if same-species fungal isolates differed in their effect.
3. An experiment was performed as a first test of the effect of intra-specific genetic variation in the mycorrhizal fungus Glomus etunicatum on mirid nymphal population structure, dynamics, and growth rate.
4. Mirid nymphal populations were lower on mycorrhizal fungal-infected plants. Population size, however, did not differ between the mycorrhizal isolates. While no statistical difference in population between isolates was found, one isolate consistently had 1.7–2.4 times lower mirid populations compared with the controls, indicating that the magnitude of effect is different between mycorrhizal isolates.
5. The significantly negative effect of AMF on mirid populations likely resulted from AMF-induced changes in plant quality (e.g. increased defence). This study lends further support to recent demonstrations that below-ground symbionts significantly influence above-ground processes. In addition, mycorrhizal fungi can affect insect population structure, which may have consequences for future herbivory.     

In vitro culture of tobacco callus on medium containing peptone and phytate leads to growth improvement and higher genetic stability

Growth and genetic stability of Nicotiana tabacum L. callus were strongly improved by replacing the inorganic nitrogen and phosphorus of the Murashige and Skoog's medium by a soybean peptone and phytate, respectively. Cell proliferation after subcultivation on the modified medium was highly stimulated as evidenced by a strong biomass increase; this improvement was mainly due to the organic N source. In addition, while calluses grown under standard conditions displayed various cell sizes and DNA contents, subcultivation on the modified medium led to homogeneous cell size distribution and stable 4C–8C DNA contents through several subcultures. This improved genetic stability was due to replacement of inorganic P by phytate, provided the presence of peptone. Such new media composition could be useful for slow-growing cell suspensions or calluses.     

Modulation of the venation pattern of cotyledons of transgenic tobacco for the tumorigenic <Emphasis Type="Italic">6b</Emphasis> gene of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> AKE10

Neoplastic plant-tissue formation, termed crown gall disease, is induced on infection with Agrobacterium tumefaciens. The tumorous tissues develop an extensive vascular system, with a venation pattern distinct from that of native host plants. We report here that the plant-tumorigenic 6b gene of the A. tumefaciens strain AKE10 is capable of inducing extensive vein formation in transgenic tobacco seedlings with distinct pattern formation. Unlike the wild-type cotyledons, transgenic cotyledons had wavy and striate veins depending on the extent of severity of leaf morphology. Graph analysis of the transgenic cotyledonous vein patterns revealed an increase in the number of branch points of veins, end-points of veins, and areas surrounded by the veins. Histological analysis showed abnormal tissue growth on the abaxial side of the cotyledon blades and continual formation of adventitious veins. These adventitiously formed veins included inverted dorso-ventrality and formation of a radial axis.     

Endophytic colonisation of tobacco,corn, wheat and soybeans by the fungal entomopathogen Beauveria bassiana (Ascomycota,Hypocreales)

We demonstrate the effectiveness of three inoculation methods (foliar spray, seed immersion and root immersion) in establishing fungal the entomopathogen Beauveria bassiana as an endophyte in tobacco, corn, wheat and soybean. Colonisation of leaves by B. bassiana was assessed 7, 14, 21 and 28 days post-inoculation. There were significant differences (p < 0.001) in endophytic colonisation among the different inoculation techniques.     

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non‐human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as ‘category A’ pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost‐effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C‐terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co‐expression of the GP1‐heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size‐exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti‐Ebola virus antibody production at levels comparable to those obtained with a GP1 virus‐like particle. These results show excellent potential for a plant‐expressed EIC as a human vaccine.     

Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.     

Gene activation in plastids by the CRE site-specific recombinase

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.