Selection and validation of reference genes for RT-qPCR indicates that juice of sugarcane varieties modulate the expression of C metabolism genes in the endophytic diazotrophic Herbaspirillum rubrisubalbicans strain HCC103.

Quantitative reverse transcription PCR (RT-qPCR) is an important tool for evaluating gene expression. However, this technique requires that specific internal normalizing genes be identified for different experimental conditions. To date, no internal normalizing genes are available for validation of data analyses for Herbaspirillum rubrisubalbicans strain HCC103, an endophyte that is part of the sugarcane consortium inoculant. This work seeks to identify and evaluate suitable reference genes for gene expression studies in HCC103 grown until middle log phase in sugarcane juice obtained from four sugarcane varieties or media with three different carbon sources. The mRNA levels of five candidate genes (rpoA, gyrA, dnaG, recA and gmK) and seven target genes involved in carbon metabolism (acnA, fbp, galE, suhB, wcaA, ORF_0127.0101 and _0127.0123) were quantified by RT-qPCR. Analysis of expression stability of these genes was carried out using geNorm and Normfinder software. The results indicated that the HCC103 dnaG and gyrA genes are the most stable and showed adequate relative expression level changes among the different sugarcane juices. The highest expression level was seen for ORF_0127.0101, which encodes a sugar transporter, in juice from sugarcane variety RB867515 and glucose as the carbon source. The suhB gene, encoding SuhB inositol monophosphatase, had a higher relative expression level on 0.5% glucose, 100% sugarcane juice from variety RB867515 and 0.5% aconitate. Together the results suggest that dnaG and gyrA genes are suitable as reference genes for RT-qPCR analysis of strain HCC103 and that juice from different sugarcane varieties modulates the expression of key genes involved in carbon metabolism.

Selection and evaluation of reference genes for RT-qPCR expression studies on Burkholderia tropica strain Ppe8, a sugarcane-associated diazotrophic bacterium grown with different carbon sources or sugarcane juice.

Among the members of the genus Burkholderia, Burkholderia tropica has the ability to fix nitrogen and promote sugarcane plant growth as well as act as a biological control agent. There is little information about how this bacterium metabolizes carbohydrates as well as those carbon sources found in the sugarcane juice that accumulates in stems during plant growth. Reverse transcription quantitative PCR (RT-qPCR) can be used to evaluate changes in gene expression during bacterial growth on different carbon sources. Here we tested the expression of six reference genes, lpxC, gyrB, recA, rpoA, rpoB, and rpoD, when cells were grown with glucose, fructose, sucrose, mannitol, aconitic acid, and sugarcane juice as carbon sources. The lpxC, gyrB, and recA were selected as the most stable reference genes based on geNorm and NormFinder software analyses. Validation of these three reference genes during strain Ppe8 growth on the same carbon sources showed that genes involved in glycogen biosynthesis (glgA, glgB, glgC) and trehalose biosynthesis (treY and treZ) were highly expressed when Ppe8 was grown in aconitic acid relative to other carbon sources, while otsA expression (trehalose biosynthesis) was reduced with all carbon sources. In addition, the expression level of the ORF_6066 (gluconolactonase) gene was reduced on sugarcane juice. The results confirmed the stability of the three selected reference genes (lpxC, gyrB, and recA) during the RT-qPCR and also their robustness by evaluating the relative expression of genes involved in glycogen and trehalose biosynthesis when strain Ppe8 was grown on different carbon sources and sugarcane juice.

Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus.

TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

Insights into the structure and role of seed-borne bacteriome during maize germination.

Seed germination events modulate microbial community composition, which ultimately influences seed-to-seedling growth performance. Here, we evaluate the germinated maize (variety SHS 5050) root bacterial community of disinfected seed (DS) and non-disinfected seed (NDS). Using a gnotobiotic system, sodium hypochlorite (1.25%; 30 min)-treated seeds showed a reduction of bacterial population size and an apparent increase of bacterial community diversity associated with a significant selective reduction of Burkholderia-related sequences. The shift in the bacterial community composition in DS negatively affects germination speed, seedling growth and reserve mobilization rates compared with NDS. A synthetic bacterial community (syncom) formed by 12 isolates (9 Burkholderia spp., 2 Bacillus spp., and 1 Staphylococcus sp.) obtained from natural microbiota maize seeds herein was capable of recovering germination and seedling growth when reintroduced in DS. Overall, results showed that changes in bacterial community composition and selective reduction of Burkholderia-related members' dominance interfere with germination events and the initial growth of the maize. By cultivation-dependent and -independent approaches, we deciphered seed-maize microbiome structure, bacterial niches location and bacterial taxa with relevant roles in seedling growth performance. A causal relationship between seed microbial community succession and germination performance opens opportunities in seed technologies to build-up microbial communities to boost plant growth and health.