Identification of botanicals using molecular biotechnology.

Book Reviews

Fungal symbionts commonly occur in plants influencing host growth, physiology, and ecology (Carlile et al., 2001). However, while whole-plant growth responses to biotrophic fungi are readily demonstrated, it has been much more difficult to identify and detect the physiological mechanisms responsible. Previous work on the clonal grass Glyceria striata has revealed that the systemic fungal endophyte Epichloë glyceriae has a positive effect on clonal growth of its host (Pan & Clay, 2002; 2003). The latest study from these authors, in this issue (pp. 467- 475), now suggests that increased carbon movement in hosts infected by E. glyceriae may function as one mechanism by which endophytic fungi could increase plant growth. Given the widespread distribution of both clonal plants and symbiotic fungi, this research will have implications for our understanding of the ecology and evolution of fungus-plant associations in natural communities.

Medicinal plants are frequently used nonspecifically to improve body functions, especially when a reduction of muscle function, cognition or sexual performance, or resistance to infections is perceived. In such cases, people often turn to tonic, rejuvenating, and restorative plants. These plants deserve more attention and scrutiny to find scientific evidence to support their popular use. In modern medicine, the adaptogen term was coined for the class of compounds that is capable of increasing nonspecific resistance of the organism by improving its ability to adapt to environmental factors and stressor agents. Adaptogen plants can also be used by healthy individuals to improve cognitive and physical performance or as geriatric agents to prevent or minimize physical and cognitive deficits. Through efficacy and safety evidence, plants known as aphrodisiacs or sexual, energizing, or rejuvenating stimulants or that are indicated for weakness or impaired memory could be regarded as potential new herbal adaptogens.

Vaccination of animals has been used for centuries and is generally considered the most cost-effective and sustainable method of disease control and prevention. About twenty-five years ago, vaccines were in a inactive form or live attenuated organisms and often were not very effective. Advances in molecular biology and biotechnology have made it possible to develop new vaccines and therapeutic targes. Plant expression system has been demonstrated to be a promising platform for production of a variety of recombinant proteins such as vaccines, antibodies, therapeutic proteins, human and industrial enzymes, toxins etc. for health, agricultural and industrial applications. Although plant produced products are already available and licensed for human use, however, there are currently no plant-based vaccines on the market for animal use other than the Newcastle poultry vaccine. This is probably explained by relatively high cost of plant produced recombinant protein based vaccines for animal use. Therefore, the development of inexpensive and affordable plant-based vaccines and their formulation is very important for the production of economical animal vaccines. In this review, (1) different expression systems, (2) the history of plant-based expression systems, (3) different types of vaccines, and(4) plant-based animal vaccine production in plants are discussed. We also discussed the advantages of plants in the development of veterinary vaccines and new developed strategies that can lead to the production of cost-effective, stable and highly immunogenic veterinary vaccines.

Vegetable protein synthesis systems for industry, medicine, and research are becoming increasingly popular. The technology of protein production in plants has certain advantages, compared with the expression systems of bacteria and yeast. The rich variety of promoters, regulatory elements, affinity tags, and fusion partners that are used in molecular biology and plant biotechnology can create hybrid genetic constructs adapted to the solution of various tasks associated with protein synthesis and purification. New methods of modification of plant systems are being developed for the synthesis of functionally active human proteins whose structure is close to the natural analogues. This review shows current approaches to increase the yield of the target protein, facilitating the procedures of its isolation and purification and preventing degradation.

The application of molecular biology to the study of human malignancies has led to tremendous gains in our understanding of their pathogenesis. Although their practical applications are still somewhat limited at this point, the use of molecular diagnostic tools is likely to grow at a very rapid rate as newer and more accurate prognostic markers are identified. The availability of reliable prognostic markers should allow earlier intervention in patients with aggressive disease but exhibiting only limited extent of disease at the time of initial diagnosis. Early intervention in such cases could realistically increase the probability of cure, since highly aggressive tumor cells are more likely to be eliminated by early institution of cytotoxic chemotherapy (4). The p53 tumor suppressor gene clearly represents the most promising potential prognostic marker at present, because of both the multiple phenotypic alterations caused by different p53 mutations and the high frequency of p53 mutations which have been observed in a variety of human cancers. Other prognostic markers related to oncogenes and tumor suppressor genes are almost certain to follow. Validation of new prognostic markers requires a knowledge of both histopathologic diagnostic criteria as well as the consequences for the patient of each diagnosis. There is bound to be some "shake-out" in the field of molecular diagnostics just as there was with other recently introduced techniques such as immunohistochemistry and flow cytometry which were found to provide additional useful information for some tumors and not for others. Since the clinical-pathologic studies needed for verification of putative prognostic markers require relatively long periods of follow up, progress in this area will almost certainly lag behind the ability of molecular biologists to identify new and potentially useful prognostic markers. Our collective ability to reap tangible gains in the clinical arena from our heavy investments in molecular biology and biotechnology depends to a large extent on open channels of communication between clinical and basic scientists. As our ever-increasing insights into oncogenic processes spawn new diagnostic and prognostic markers, our priorities should remain focused on those areas which are inadequately addressed by current methods, and we should avoid the technological trap of devising redundant solutions which increase the expense, but not the efficiency of patient care.

Editorial and Retrospective 2008

<p><b>Phytophthora – Greek for “the plant-destroyer” – is a genus of eukaryotic microorganisms that cause devastating plant diseases worldwide. Collectively, Phytophthora species cause billions of dollars in damages to crops and native ecosystems annually. Despite these extensive impacts, Phytophthora research is challenging due to a lack of molecular biology tools. Widely used techniques such as transformation and gene deletion remain challenging to establish in Phytophthora species. However, CRISPR-Cas genome editing was recently achieved in a small number of model Phytophthora species – but these breakthroughs have yet to reach most members of the genus. The pathogen Phytophthora agathidicida is one example. P. agathidicida causes kauri dieback, a disease that threatens kauri, one of our most important tree species in New Zealand. P. agathidicida was formally named in 2015 and is therefore an emergent threat. Consequently, key molecular methods such as transformation and CRISPR-Cas genome editing have yet to be established for this pathogen.</b></p> <p>The overarching goal of this thesis was to establish molecular biology methods for the study of P. agathidicida. The first aim was to identify a suitable target for genome editing with CRISPR-Cas and design guide RNAs for the target. A putative thymidine kinase gene from the P. agathidicida isolate 3770 genome was selected as a target, and eight guide RNA sequences were designed. All of the guide RNAs successfully directed the digestion of the target gene in vitro. The second aim was to establish a method to produce and transform P. agathidicida protoplasts. An optimised recovery medium was identified using a mock transformation method, which increased protoplast germination rates from 2.4% in a previously used medium to 15% in the new medium. Using the optimised conditions, P. agathidicida was transformed with plasmids encoding Cas nuclease-GFP fusion proteins. Transformants were both fluorescent and antibiotic resistant. The third aim was to express, purify, and characterise the putative thymidine kinase enzyme. Yeast species Pichia pastoris was used as a protein expression host, but neither protein expression nor kinase activity were consistently observed. </p> <p>In this thesis, P. agathidicida was successfully transformed for the first time. Transformation is a huge advancement and will greatly accelerate molecular biology research in P. agathidicida for years to come. In particular, transformation is a key step towards CRISPR-Cas genome editing in P. agathidicida. CRISPR-Cas will not only provide insights into its fundamental biology, but may guide the development of new control strategies for this devastating pathogen.</p>

Growth and productivity of crop plants worldwide are often adversely affected by anthropogenic and natural stresses. Both biotic and abiotic stresses may impact future food security and sustainability; global climate change will only exacerbate the threat. Nearly all stresses induce ethylene production in plants, which is detrimental to their growth and survival when present at higher concentrations. Consequently, management of ethylene production in plants is becoming an attractive option for countering the stress hormone and its effect on crop yield and productivity. In plants, ACC (1-aminocyclopropane-1-carboxylate) serves as a precursor for ethylene production. Soil microorganisms and root-associated plant growth promoting rhizobacteria (PGPR) that possess ACC deaminase activity regulate growth and development of plants under harsh environmental conditions by limiting ethylene levels in plants; this enzyme is, therefore, often designated as a "stress modulator." TheACC deaminase enzyme, encoded by the AcdS gene, is tightly controlled and regulated depending upon environmental conditions. Gene regulatory components of AcdS are made up of the LRP protein-coding regulatory gene and other regulatory components that are activated via distinct mechanisms under aerobic and anaerobic conditions. ACC deaminase-positive PGPR strains can intensively promote growth and development of crops being cultivated under abiotic stresses including salt stress, water deficit, waterlogging, temperature extremes, and presence of heavy metals, pesticides and other organic contaminants. Strategies for combating environmental stresses in plants, and improving growth by introducing the acdS gene into crop plants via bacteria, have been investigated. In the recent past, some rapid methods and cutting-edge technologies based on molecular biotechnology and omics approaches involving proteomics, transcriptomics, metagenomics, and next generation sequencing (NGS) have been proposed to reveal the variety and potential of ACC deaminase-producing PGPR that thrive under external stresses. Multiple stress-tolerant ACC deaminase-producing PGPR strains have demonstrated great promise in providing plant resistance/tolerance to various stressors and, therefore, it could be advantageous over other soil/plant microbiome that can flourish under stressed environments.

Introduction to the special issue of “Advances in Molecular Imaging”

The rhizosphere microbiome is crucial for plant growth and health, providing defense against plethora of surrounding potential pathogens, improving crops’ nutrient acquisition, and aiding in withstanding series of abiotic stresses. Exchange of resources between plants and their associated soil environment is supported by a pivotal interface termed as rhizosphere. Plants being sessile are incessantly exposed to a diverse array of abiotic and biotic stresses under natural conditions, thereby acting as major bottlenecks to hinder their growth and production. However, it is possible to engineer plant rhizosphere microbiome as revealed by the recent advances in research in context to the plant–microbe interactions. Therefore, shaping of rhizosphere microbiome for developing promising strategies can be vital in relation to plant productivity. Furthermore, exploring the structure and dynamics of plant rhizosphere microbiome represents an exciting frontier of research to protect plants from potential phytopathogens in a sustainable manner. Advances in molecular tools are beneficial to unravel the concepts of plant–microbe association, which could act as a key driver in drafting the future “biofertilizers.” Owing to the current challenges in crop production, there is an urgent need to understand plant–microbiome interactions in the rhizosphere to bring microbiome-based strategies for incorporating beneficial resident microbial communities into practice. The present chapter uncovers the concepts of plant rhizosphere microbiome, its diversity, abundance, composition, and interplay with the plants. Additionally, elucidation of plant rhizosphere microbiome engineering and various factors and techniques involved therein for enhancing plant productivity have been presented, thereby helping meet food requirements of exponentially growing global population.KeywordsRhizosphere microbiomeHolobiontRoot exudationRhizosphere engineeringBioinformatics

Molecular identification of the Sporothrix schenckii complex

Composting is an aerobic biological process in which solid organic matter is degraded by microorganisms. The microbiology of composting has been of interest for decades, and microbes in composting have been characterized in many types of composing processes using traditional culture-based methods. In recent years, an immense diversity of bacteria, archaea, and fungi has been found to occupy many different habitats using culture-independent molecular biological methods. Molecular methods which can detect both the culturable and non-culturable fractions of the microbial community are under constant development. In this chapter, several new molecular tools for characterising the microbes present in different composting processes are described, and the advantages and limitations of the application of these methods in studying composting microbiology are discussed.

Grass species are often included during restoration to reinstate feedbacks with fire. In biodiverse pine savannas of the southeastern United States, wiregrass (Aristida beyrichiana) is the preferred candidate for restoring low‐intensity fires through the understory. Less common but locally co‐dominant in dry pine savannas, pineywoods dropseed (Sporobolus junceus) is another bunchgrass candidate for restoration. If two bunchgrass species associate with different suites of plants in the understory, restoring both could support a twofold goal of restoring fire regimes and plant biodiversity. To guide restoration goals, we tested the hypothesis that in natural (i.e. not recently restored) dry pine savannas in North Florida where wiregrass and dropseed are co‐dominant, they are associated with different understory plant species richness, occurrence, and abundance under or adjacent to their canopies. Species richness of associated plants did not differ between bunchgrass species or with distance from bunchgrass (i.e. under vs. adjacent). Some associated plant species, however, were more likely to occur and had greater abundance with one or the other bunchgrass species irrespective of distance. Our results suggest that including more than one bunchgrass species in restoration plantings could increase dry pine savanna biodiversity while also reinstating plant‐fire feedbacks.

Here we review the application of molecular biological approaches to mineral precipitation in modern marine microbialites. The review focuses on the nearly two decades of nucleotide sequencing studies of the microbialites of Shark Bay, Australia; and The Bahamas. Molecular methods have successfully characterized the overall community composition of mats, pinpointed microbes involved in key metabolisms, and revealed patterns in the distributions of microbial groups and functional genes. Molecular tools have become widely accessible, and we can now aim to establish firmer links between microbes and mineralization. Two promising future directions include “zooming in” to assess the roles of specific organisms, microbial groups, and surfaces in carbonate biomineralization and “zooming out” to consider broader spans of space and time. A middle ground between the two can include model systems that contain representatives of important microbial groups, processes, and metabolisms in mats and simplify hypothesis testing. These directions will benefit from expanding reference datasets of marine microbes and enzymes and enrichments of representative microbes from mats. Such applications of molecular tools should improve our ability to interpret ancient and modern microbialites and increase the utility of these rocks as long-term recorders of microbial processes and environmental chemistry.