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.

Identification of proteins in susceptible and resistant Brassica oleracea responsive to Xanthomonas campestris pv. campestris infection.

UNLABELLED: Cruciferous plants are important edible vegetables widely consumed around the world, including cabbage, cauli-flower and broccoli. The main disease that affects crucifer plants is black rot, caused by Xanthomonas campestris pv. campestris (Xcc). In order to better understand this specific plant-pathogen interaction, proteins responsive to Xcc infection in resistant (União) and susceptible (Kenzan) Brassica oleracea cultivars were investigated by 2-DE followed by mass spectrometry. A total of 47 variable spots were identified and revealed that in the susceptible interaction there is a clear reduction in the abundance of proteins involved in energetic metabolism and defense. It was interesting to observe that in the resistant interaction, these proteins showed an opposite behavior. Based on our results, we conclude that resistance is correlated with the ability of the plant to keep sufficient photosynthesis metabolism activity to provide energy supplies necessary for an active defense. As a follow-up study, qRT-PCR analysis of selected genes was performed and revealed that most genes showed an up-regulation trend from 5 to 15days after inoculation (DAI), showing highest transcript levels at 15DAI. These results revealed the gradual accumulation of transcripts providing a more detailed view of the changes occurring during different stages of the plant-pathogen interaction. BIOLOGICAL SIGNIFICANCE: In this study we have compared cultivars of Brassica oleracea (cabbage), susceptible and resistant to black rot, by using the classical 2-DE approach. We have found that resistance is correlated with the ability of the plant to keep sufficient photosynthesis metabolism activity to provide energy supplies necessary for an active defense.

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.

Microbiological functioning, diversity, and structure of bacterial communities in ultramafic soils from a tropical savanna.

Ultramafic soils are characterized by high levels of metals, and have been studied because of their geochemistry and its relation to their biological component. This study evaluated soil microbiological functioning (SMF), richness, diversity, and structure of bacterial communities from two ultramafic soils and from a non-ultramafic soil in the Brazilian Cerrado, a tropical savanna. SMF was represented according to simultaneous analysis of microbial biomass C (MBC) and activities of the enzymes ß-glucosidase, acid phosphomonoesterase and arylsulfatase, linked to the C, P and S cycles. Bacterial community diversity and structure were studied by sequencing of 16S rRNA gene clone libraries. MBC and enzyme activities were not affected by high Ni contents. Changes in SMF were more related to the organic matter content of soils (SOM) than to their available Ni. Phylogeny-based methods detected qualitative and quantitative differences in pairwise comparisons of bacterial community structures of the three sites. However, no correlations between community structure differences and SOM or SMF were detected. We believe this work presents benchmark information on SMF, diversity, and structure of bacterial communities for a unique type of environment within the Cerrado biome.

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.

Molecular cloning of a cysteine proteinase cDNA from the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.