Differential expression of genes potentially related to the callogenesis and in situ hybridization of SERK gene in macaw palm (Acrocomia aculeata Jacq.) Lodd. ex Mart.

For the purpose of understanding the molecular processes triggered during callus formation in macaw palm, the expression of seven genes potentially involved in this process, identified in previous studies and from the literature, was investigated by RT-qPCR. In addition, in situ hybridization of the SERK gene was performed. Leaf tissues from adult plants from two macaw palm accession were inoculated in a medium combined with Picloram at a concentration of 450 µM to induce callus. The expression analysis was performed from leaf samples from two accessions of different origins (Municipalities of Tiros, MG, and Buriti Vermelho, DF, Brazil), which are characterized as non-responsive (NR) and responsive (R), respectively. The material was collected before callus induction (0 DAI, initial day) and 120 days after callus induction (120 DAI). Genes related to development (SERK, OASA, EF1, ANN1) and stress (LEA, CAT2, and MDAR5) were evaluated. The results obtained showed that all the genes involved with the development had their expressions downregulated at 0 DAI when the accession R was compared with the accession NR. On the other hand, it was possible to observe that these genes were upregulated at 120 DAI. The LEA stress gene showed a tendency to increase expression in the NR accession, while the R accession showed decreased expression and the CAT2 and MDAR5 genes showed upregulation in both accessions. In situ hybridization showed SERK transcripts in the vascular bundles, indicating the expression of SERK in this region, in addition to its expression in calluses. The results obtained in this study support our hypothesis that the regulation of genes involved in the control of oxidative stress and development is crucial for the formation of calluses in macaw palm.

A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

Somatic embryogenesis as an alternative for in vitro multiplication of Butia odorata from mature zygotic embryos.

Butia odorata is a palm native to southern Brazil and Uruguay, not domesticated, much appreciated for its fruits and economic potential. However, the extractivism and the difficulty of propagation have led to the decline of natural populations. The objective of this work was to prove the possibility of induction of somatic embryogenesis in B. odorata. Mature zygotic embryos were induced in two media, MS and Y3, combined with auxin 2,4-D and picloram in five concentrations (2,4-D: 0, 361.99, 452.49, 542.99 and 633.48 µM/L, picloram: 0, 50, 150, 300 and 450 µM/L). The results promising during induction with the formation of embryogenic calli and somatic embryos, however the regeneration of them was not efficient, this may be due to the occurrence of somatic embryos fused during its development. The roots were formed, but the aerial part remained molten, not completing its development. Auxin picloram and Y3 medium provided the most adequate conditions for calogenesis, formation of embryogenic callus and somatic embryos, with concentrations of 150, 300 and 450 µM/L. This is the first description of somatic embryogenesis in B. odorata that will serve as the basis for future research and adjustments of the methodology proposed here.

Plin-amiR, a pre-microRNA-based technology for controlling herbivorous insect pests.

The cotton bollworm, Helicoverpa armigera, is a major insect pest for a wide range of agricultural crops. It causes significant yield loss through feeding damage and by increasing the crop's vulnerability to bacterial and fungal infections. Although expression of Bacillus thuringiensis (Bt) toxins in transgenic crops has been very successful in protecting against insect pests, including H. armigera, field-evolved resistance has occurred in multiple species. To manage resistant populations, new protection strategies must be continuously developed. Trans-kingdom RNA interference (TK-RNAi) is a promising method for controlling herbivorous pests. TK-RNAi is based on delivering dsRNA or hairpin RNA containing essential insect gene sequences to the feeding insect. The ingested molecules are processed by the insect's RNAi machinery and guide it to silence the target genes. Recently, TK-RNAi delivery has been enhanced by expressing the ds- or hpRNAs in the chloroplast. This compartmentalizes the duplexed RNA away from the plant's RNAi machinery, ensuring that it is delivered in an unprocessed form to the insect. Here, we report another alternative approach for delivering precursor anti-insect RNA in plants. Insect pre-microRNA (pre-miR) transcripts were modified to contain artificial microRNAs (amiRs), targeting insect genes, and expressed in transgenic Nicotiana benthamiana plants. These modified pre-miRs remained largely unprocessed in the plants, and H. armigera feeding on leaves from these plants had increased mortality, developmental abnormalities and delayed growth rates. This shows that plant-expressed insect pre-amiRs (plin-amiRs) are a new strategy of protecting plants against herbivorous insects.

Transcriptome Analysis of Paraburkholderia phymatum under Nitrogen Starvation and during Symbiosis with Phaseolus Vulgaris.

Paraburkholderia phymatum belongs to the ß-subclass of proteobacteria. It has recently been shown to be able to nodulate and fix nitrogen in symbiosis with several mimosoid and papilionoid legumes. In contrast to the symbiosis of legumes with α-proteobacteria, very little is known about the molecular determinants underlying the successful establishment of this mutualistic relationship with ß-proteobacteria. In this study, we performed an RNA-sequencing (RNA-seq) analysis of free-living P. phymatum growing under nitrogen-replete and -limited conditions, the latter partially mimicking the situation in nitrogen-deprived soils. Among the genes upregulated under nitrogen limitation, we found genes involved in exopolysaccharides production and in motility, two traits relevant for plant root infection. Next, RNA-seq data of P. phymatum grown under free-living conditions and from symbiotic root nodules of Phaseolus vulgaris (common bean) were generated and compared. Among the genes highly upregulated during symbiosis, we identified-besides the nif gene cluster-an operon encoding a potential cytochrome o ubiquinol oxidase (Bphy_3646-49). Bean root nodules induced by a cyoB mutant strain showed reduced nitrogenase and nitrogen fixation abilities, suggesting an important role of the cytochrome for respiration inside the nodule. The analysis of mutant strains for the RNA polymerase transcription factor RpoN (σ54) and its activator NifA indicated that-similar to the situation in α-rhizobia-P. phymatum RpoN and NifA are key regulators during symbiosis with P. vulgaris.

Competition Experiments for Legume Infection Identify Burkholderia phymatum as a Highly Competitive ß-Rhizobium.

Members of the genus Burkholderia (ß-proteobacteria) have only recently been shown to be able to establish a nitrogen-fixing symbiosis with several legumes, which is why they are also referred to as ß-rhizobia. Therefore, very little is known about the competitiveness of these species to nodulate different legume host plants. In this study, we tested the competitiveness of several Burkholderia type strains (B. diazotrophica, B. mimosarum, B. phymatum, B. sabiae, B. symbiotica and B. tuberum) to nodulate four legumes (Phaseolus vulgaris, Macroptilium atropurpureum, Vigna unguiculata and Mimosa pudica) under our closely defined growth conditions. The assessment of nodule occupancy of these species on different legume host plants revealed that B. phymatum was the most competitive strain in the three papilionoid legumes (bean, cowpea and siratro), while B. mimosarum outcompeted the other strains in mimosa. The analysis of phenotypes known to play a role in nodulation competitiveness (motility, exopolysaccharide production) and additional in vitro competition assays among ß-rhizobial strains suggested that B. phymatum has the potential to be a very competitive legume symbiont.

Mutations in Two Paraburkholderia phymatum Type VI Secretion Systems Cause Reduced Fitness in Interbacterial Competition.

Paraburkholderia phymatum is a highly effective microsymbiont of Mimosa spp. and has also been shown to nodulate papilionoid legumes. P. phymatum was found to be highly competitive both in a natural environment as well as under controlled test conditions and is more competitive for nodulation over other α- and ß-rhizobial strains in a variety of different plant hosts. In order to elucidate the factors that make this bacterium highly competitive for legume infection, we here characterized the type VI secretion system (T6SS) clusters of P. phymatum. T6SSs have been shown to function as a contact-dependent injection system for both bacterial and eukaryotic cells. We identified two T6SS clusters in the genome, created respective mutant strains and showed that they are defective in biofilm formation and in interbacterial competition in vitro. While the T6SS mutants were as efficient as the wild-type in nodulating the non-cognate host Vigna unguiculata, the mutants were less competitive in in planta competition assays, suggesting that the T6SS is one of the factors responsible for the success of P. phymatum in infecting legumes by directly inhibiting competitors.

Alternative nitrogenase and pseudogenes: unique features of the Paenibacillus riograndensis nitrogen fixation system.

Biological nitrogen fixation (BNF) is a tightly regulated process that is carried out by diazotrophic microorganisms. The regulatory mechanisms of BNF-related genes are well characterized in Gram-negative models, but they are poorly understood in Gram-positive bacteria. Paenibacillus riograndensis SBR5(T) is a Gram-positive, endospore-forming facultative aerobic diazotroph. Three clusters of BNF-related genes with dissimilar phylogenetic histories were identified in the P. riograndensis genome, and no regulatory genes were recognized. P. riograndensis nifH2 was considered inactive based on transcript and promoter analyses, whereas transcripts of nifH1 and anfH were induced upon nitrogen-limited conditions. The functionality of the alternative nitrogenase system was also validated by enzymatic activity analysis. Fragments upstream of the two active clusters seem to harbor primary functional promoter sequences, producing a constitutive expression pattern in Escherichia coli. Sequences upstream of the anf genes were not recognized by this heterologous host, indicating a very distinct promoter pattern. These results shed light upon the evolutionary history of nitrogen fixation genes in this Gram-positive bacterium and highlight the presence of novel regulatory elements that are yet to be described.

Changes in root bacterial communities associated to two different development stages of canola (Brassica napus L. var oleifera) evaluated through next-generation sequencing technology.

Crop production may benefit from plant growth-promoting bacteria. The knowledge on bacterial communities is indispensable in agricultural systems that intend to apply beneficial bacteria to improve plant health and production of crops such as canola. In this work, the diversity of root bacterial communities associated to two different developmental phases of canola (Brassica napus L.) plants was assessed through the application of new generation sequencing technology. Total bacterial DNA was extracted from root samples from two different growth states of canola (rosette and flowering). It could be shown how bacterial communities inside the roots changed with the growing stage of the canola plants. There were differences in the abundance of the genera, family, and even the phyla identified for each sample. While in both root samples Proteobacteria was the most common phylum, at the rosette stage, the most common bacteria belonged to the family Pseudomonadaceae and the genus Pseudomonas, and in the flowering stage, the Xanthomonadaceae family and the genus Xanthomonas dominated the community. This implies in a switch in the predominant bacteria in the different developmental stages of the plant, suggesting that the plant itself interferes with the associated microbial community.

Genome sequence of the diazotrophic Gram-positive rhizobacterium Paenibacillus riograndensis SBR5(T).

Paenibacillus riograndensis SBR5(T), a nitrogen-fixing Gram-positive rhizobacterium isolated from a wheat field in the south of Brazil, has a great potential for agricultural applications due to its plant growth promotion effects. Here we present the draft genome sequence of P. riograndensis SBR5(T). Its 7.37-Mb genome encodes determinants of the diazotrophic lifestyle and plant growth promotion, such as nitrogen fixation, antibiotic resistance, nitrate utilization, and iron uptake.

Roles of flavonoids and the transcriptional regulator TtsI in the activation of the type III secretion system of Bradyrhizobium elkanii SEMIA587.

Bradyrhizobium elkanii SEMIA587 is a symbiotic nitrogen-fixing bacterium of the group commonly called rhizobia, which induce nodule formation in legumes, and is widely used in Brazilian commercial inoculants of soybean. In response to flavonoid compounds released by plant roots, besides Nod factors, other molecular signals are secreted by rhizobia, such as proteins secreted by type III secretion systems (T3SSs). Rhizobial T3SSs are activated by the transcription regulator TtsI, which binds to sequences present in the promoter regions of T3SS genes via a conserved sequence called the tts box. To study the role of the T3SS of B. elkanii SEMIA587, ttsI was mutated. Protein secretion and flavonoid induction analysis, as well as nodulation tests, were performed with the wild-type and mutant strains. The results obtained showed that B. elkanii SEMIA587 secretes at least two proteins (NopA and NopL, known rhizobial T3SS substrates) after genistein induction, whilst supernatants of the ttsI mutant did not contain these Nops. Unusually for rhizobia, the promoter region of the B. elkanii SEMIA587 ttsI gene contains a tts box, which is responsive to flavonoid induction and to which TtsI can bind. Nodulation tests performed with three different leguminous plants showed that the B. elkanii SEMIA587 ttsI mutant displays host-dependent characteristics; in particular, nodulation of two soybean cultivars, Peking and EMBRAPA 48, was more efficient when TtsI of B. elkanii was functional.

Relationship between in vitro enhanced nitrogenase activity of an Azospirillum brasilense Sp7 mutant and its growth-promoting activities in situ.

In this work, we further analyzed an Azospirillum brasilense Sp7 mutant (Sp7::Tn5-33) showing a pleiotrophic phenotype due to a Tn5 insertion into an open reading frame of 840 bp (orf280). The deduced amino acid sequence of this region has high similarity to a family of universal stress proteins. Because the most interesting property exhibited by the Sp7::Tn5-33 mutant was an enhanced in vitro nitrogen fixation activity, we addressed the question of whether it could benefit the host plant. We found that the increased nitrogenase activity at the free-living state of the mutant bacterium was correlated with an increased production of the nitrogenase reductase protein (NifH), in amounts approximately 1.5 times higher than the wild type. The mutant strain exhibited the same level of auxin production and the same colonization pattern of wheat roots as the wild type. We also observed that Sp7::Tn5-33 increased the total plant dry weight, although the N content did not differ significantly between wheat plants inoculated with mutant or wild-type strains.