Functional characterization of the pUceS8.3 promoter and its potential use for ectopic gene overexpression.

MAIN CONCLUSION: The pUceS8.3 is a constitutive gene promoter with potential for ectopic and strong genes overexpression or active biomolecules in plant tissues attacked by pests, including nematode-induced giant cells or galls. Soybean (Glycine max) is one of the most important agricultural commodities worldwide and a major protein and oil source. Herein, we identified the soybean ubiquitin-conjugating (E2) enzyme gene (GmUBC4; Glyma.18G216000), which is significantly upregulated in response to Anticarsia gemmatalis attack and Meloidogyne incognita-induced galls during plant parasitism by plant nematode. The GmUBC4 promoter sequence and its different modules were functionally characterized in silico and in planta using transgenic Arabidopsis thaliana and G. max lines. Its full-length transcriptional regulatory region (promoter and 5´-UTR sequences, named pUceS8.3 promoter) was able to drive higher levels of uidA (ß-glucuronidase) gene expression in different tissues of transgenic A. thaliana lines compared to its three shortened modules and the p35SdAMV promoter. Notably, higher ß-glucuronidase (GUS) enzymatic activity was shown in M. incognita-induced giant cells when the full pUceS8.3 promoter drove the expression of this reporter gene. Furthermore, nematode-specific dsRNA molecules were successfully overexpressed under the control of the pUceS8.3 promoter in transgenic soybean lines. The RNAi gene construct used here was designed to post-transcriptionally downregulate the previously characterized pre-mRNA splicing factor genes from Heterodera glycines and M. incognita. A total of six transgenic soybean lines containing RNAi gene construct were selected for molecular characterization after infection with M. incognita pre-parasitic second-stage (ppJ2) nematodes. A strong reduction in the egg number produced by M. incognita after parasitism was observed in those transgenic soybean lines, ranging from 71 to 92% compared to wild-type control plants. The present data demonstrated that pUceS8.3 is a gene promoter capable of effectively driving dsRNA overexpression in nematode-induced giant cells of transgenic soybean lines and can be successfully applied as an important biotechnological asset to generate transgenic crops with improved resistance to root-knot nematodes as well as other pests.

Cloning and characterization of novel cyclotides genes from South American plants.

Cyclotides are multifunctional plant cyclic peptides containing 28-37 amino acid residues and a pattern of three disulfide bridges, forming a motif known as the cyclic cystine knot. Due to their high biotechnological potential, the sequencing and characterization of cyclotide genes are crucial not only for cloning and establishing heterologous expression strategies, but also to understand local plant evolution in the context of host-pathogen relationships. Here, two species from the Brazilian Cerrado, Palicourea rigida (Rubiaceae) and Pombalia lanata (A.St.-Hil.) Paula-Souza (Violaceae), were used for cloning and characterizing novel cyclotide genes. Using 3' and 5' RACE PCR and sequencing, two full cDNAs, named parigidin-br2 (P. rigida) and hyla-br1 (P. lanata), were isolated and shown to have similar genetic structures to other cyclotides. Both contained the conserved ER-signal domain, N-terminal prodomain, mature cyclotide domain and a C-terminal region. Genomic sequencing of parigidin-br2 revealed two different gene copies: one intronless allele and one presenting a rare 131-bp intron. In contrast, genomic sequencing of hyla-br1 revealed an intronless gene-a common characteristic of members of the Violaceae family. Parigidin-br2 5' and 3' UTRs showed the presence of 12 putative candidate sites for binding of regulatory proteins, suggesting that the flanking and intronic regions of the parigidin-br2 gene must play important roles in transcriptional rates and in the regulation of temporal and spatial gene expression. The high degree of genetic similarity and structural organization among the cyclotide genes isolated in the present study from the Brazilian Cerrado and other well-characterized plant cyclotides may contribute to a better understanding of cyclotide evolution.

Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism.

Critical aspects to be considered prior to large-scale production of peptides.

Numerous peptides are available on the market as therapeutic drugs for regulating tumor growth, microorganism proliferation, immune response and/or metabolic disorders. Peptides are produced either by chemical synthesis or heterologous expression. Independent of the method chosen, there are challenges to transferring its production from the bench (~mg/L) to the industrial (~g/L) scale. Thus, the main scale-up pitfalls for the two methods of peptide production are reviewed here, including the advantages of each. Moreover, there will be a special focus on the main challenges for large-scale, heterologous production systems. Peptides that are currently available on the market are also described with an emphasis on how their process optimization has been designed in order to develop a cost-effective product.

Validation of reference genes aiming accurate normalization of qPCR data in soybean upon nematode parasitism and insect attack.

BACKGROUND: Soybean pathogens and pests reduce grain production worldwide. Biotic interaction cause extensive changes in plant gene expression profile and the data produced by functional genomics studies need validation, usually done by quantitative PCR. Nevertheless, this technique relies on accurate normalization which, in turn, depends upon the proper selection of stable reference genes for each experimental condition. To date, only a few studies were performed to validate reference genes in soybean subjected to biotic stress. Here, we report reference genes validation in soybean during root-knot nematode (Meloidogyne incognita) parasitism and velvetbean caterpillar (Anticarsia gemmatalis) attack. FINDINGS: The expression stability of nine classical reference genes (GmCYP2, GmELF1A, GmELF1B, GmACT11, GmTUB, GmTUA5, GmG6PD, GmUBC2 and GmUBC4) was evaluated using twenty-four experimental samples including different organs, developmental stages, roots infected with M. incognita and leaves attacked by A. gemmatalis. Two different algorithms (geNorm and NormFinder) were used to determine expression stability. GmCYP2 and GmUBC4 are the most stable in different organs. Considering the developmental stages, GmELF1A and GmELF1B genes are the most stable. For spatial and temporal gene expression studies, normalization may be performed using GmUBC4, GmUBC2, GmCYP2 and GmACT11 as reference genes. Our data indicate that both GmELF1A and GmTUA5 are the most stable reference genes for data normalization obtained from soybean roots infected with M. incognita, and GmCYP2 and GmELF1A are the most stable in soybean leaves infested with A. gemmatalis. CONCLUSIONS: Future expression studies using nematode infection and caterpilar infestation in soybean plant may utilize the reference gene sets reported here.

Plant biofarming: novel insights for peptide expression in heterologous systems.

Peptide expression methods have been widely studied and developed from many different biological sources. The cultivation ofprokaryotic and eukaryotic cells has proven to be efficient for the expression of foreign peptides in several heterologous systems, including bacteria, insects, yeasts, and mammals. Earlier reports brought up new insights for the improvement of expressed products to not only increase the production rate of desired peptides but also reproduce desirable post-translational modifications and even to reduce the risk of allergenicity when those products are aimed for human use. The development of bioreactor systems provided the optimization of cell growth conditions to scale up the amounts of expressed peptides. On the other hand, different cell systems and mutants provided a plethora of possible peptide modifications. Hence, in this report, we describe the many organisms and systems used for the large scale production of several macromolecules with relevance in health and agriculture. We also bring into discussion plant biofarming in the moss Physcomitrella patens and its recent adaptations, as a cost-effective and efficient approach in the production of more complex heterologous proteins, given the fact that its glycosylation pattern can be engineered to avoid allergenicity to humans (common to plant-derived glycoproteins).

Expression systems for heterologous production of antimicrobial peptides.

Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.

Expression analysis in response to drought stress in soybean: Shedding light on the regulation of metabolic pathway genes.

Metabolomics analysis of wild type Arabidopsis thaliana plants, under control and drought stress conditions revealed several metabolic pathways that are induced under water deficit. The metabolic response to drought stress is also associated with ABA dependent and independent pathways, allowing a better understanding of the molecular mechanisms in this model plant. Through combining an in silico approach and gene expression analysis by quantitative real-time PCR, the present work aims at identifying genes of soybean metabolic pathways potentially associated with water deficit. Digital expression patterns of Arabidopsis genes, which were selected based on the basis of literature reports, were evaluated under drought stress condition by Genevestigator. Genes that showed strong induction under drought stress were selected and used as bait to identify orthologs in the soybean genome. This allowed us to select 354 genes of putative soybean orthologs of 79 Arabidopsis genes belonging to 38 distinct metabolic pathways. The expression pattern of the selected genes was verified in the subtractive libraries available in the GENOSOJA project. Subsequently, 13 genes from different metabolic pathways were selected for validation by qPCR experiments. The expression of six genes was validated in plants undergoing drought stress in both pot-based and hydroponic cultivation systems. The results suggest that the metabolic response to drought stress is conserved in Arabidopsis and soybean plants.