Bidimensional Analyses of the Intra- and Extracellular Proteomes of Steroid Producer Mycobacteria.

The importance of the pathogenic mycobacteria has mainly focused the omic analyses on different aspects of their clinical significance. However, those industrially relevant mycobacteria have received less attention, even though the steroid market sales in 2021 were estimated in $56.45 billion.The extracellular proteome, due to its relevance in the sterol processing and uptake, and the intracellular proteome, because of its role in steroids bioconversion, are the core of the present chapter. Both, monodimensional gels, as preparatory analysis, and bidimensional gels as proteome analysis are described. As a proof of concept, the protein extraction methods for both sub-proteomes of Mycobacterium are described. Thus, procedures and relevant key points of these proteome analyses are fully detailed.

Advances in the control of phytopathogenic fungi that infect crops through their root system.

Productivity and economic sustainability of many herbaceous and woody crops are seriously threatened by numerous phytopathogenic fungi. While symptoms associated with phytopathogenic fungal infections of aerial parts (leaves, stems and fruits) are easily observable and therefore recognizable, allowing rapid or preventive action to control this type of infection, the effects produced by soil-borne fungi that infect plants through their root system are more difficult to detect. The fact that these fungi initiate infection and damage underground implies that the first symptoms are not as easily noticeable, and therefore both crop yield and plant survival are frequently severely compromised by the time the infection is found. In this paper we will review and discuss recent insights into plant-microbiota interactions in the root system crucial to understanding the beginning of the infectious process. We will also review different methods for diminishing and controlling the infection rate by phytopathogenic fungi penetrating through the root system including both the traditional use of biocontrol agents such as antifungal compounds as well as some new strategies that could be used because of their effective application, such as nanoparticles, virus-based nanopesticides, or inoculation of plant material with selected endophytes. We will also review the possibility of modeling and influencing the composition of the microbial population in the rhizosphere environment as a strategy for nudging the plant-microbiome interactions toward enhanced beneficial outcomes for the plant, such as controlling the infectious process.

Trans-Graft Protection Against Pierce's Disease Mediated by Transgenic Grapevine Rootstocks.

A field study showed that transgenic grapevine rootstocks can provide trans-graft-mediated protection to a wild type scion against Pierce's disease (PD) development. We individually field-tested two distinct strategies. The first expressed a chimeric antimicrobial protein (CAP) that targeted the functionality of the lipopolysaccharide (LPS) surface of Xylella fastidiosa (Xf), the causative agent of PD. The second expressed a plant polygalacturonase inhibitory protein (PGIP) that prevents PD by inhibiting breakdown of pectin present in primary cell walls. Both proteins are secreted to the apoplast and then into the xylem, where they migrate past the graft union, transiting into the xylem of the grafted scion. Transgenic Vitis vinifera cv. Thompson Seedless (TS) expressing ether CAP or PGIP were tested in the greenhouse and those lines that showed resistance to PD were grafted with wild type TS scions. Grafted grapevines were introduced into the field and tested over 7 years. Here we present data on the field evaluation of trans-graft protection using four CAP and four PGIP independent rootstock lines, compared to an untransformed rootstock. There was 30 to 95% reduction in vine mortality among CAP- and PGIP-expressing lines after three successive yearly infections with virulent Xf. Shoot tissues grafted to either CAP or PGIP transgenic rootstocks supported lower pathogen titers and showed fewer disease symptoms. Grafted plants on transgenic rootstocks also had more spring bud break following infection, more shoots, and more vigorous growth compared to those grafted to wild type rootstocks. No yield penalty was observed in the transgenic lines and some PGIP-expressing vines had enhanced yield potential. Trans-graft protection is an efficient way to protect grape scions against PD while preserving their valuable varietal genotypes and clonal properties.

Identifying Host Molecular Features Strongly Linked With Responses to Huanglongbing Disease in Citrus Leaves.

A bioinformatic analysis of previously published RNA-Seq studies on Huanglongbing (HLB) response and tolerance in leaf tissues was performed. The aim was to identify genes commonly modulated between studies and genes, pathways and gene set categories strongly associated with this devastating Citrus disease. Bioinformatic analysis of expression data of four datasets present in NCBI provided 46-68 million reads with an alignment percentage of 72.95-86.76%. Only 16 HLB-regulated genes were commonly identified between the three leaf datasets. Among them were key genes encoding proteins involved in cell wall modification such as CESA8, pectinesterase, expansin8, expansin beta 3.1, and a pectate lyase. Fourteen HLB-regulated genes were in common between all four datasets. Gene set enrichment analysis showed some different gene categories affected by HLB disease. Although sucrose and starch metabolism was highly linked with disease symptoms, different genes were significantly regulated depending on leaf growth and infection stages and experimental conditions. Histone-related transcription factors were highly affected by HLB in the analyzed RNA-Seq datasets. HLB tolerance was linked with induction of proteins involved in detoxification. Protein-protein interaction (PPI) network analysis confirmed a possible role for heat shock proteins in curbing disease progression.

Transcriptome and metabolome analysis of citrus fruit to elucidate puffing disorder.

A systems-level analysis reveals details of molecular mechanisms underlying puffing disorder in Citrus fruit. Flavedo, albedo and juice sac tissues of normal fruits and fruits displaying symptoms of puffing disorder were studied using metabolomics at three developmental stages. Microarrays were used to compare normal and puffed fruits for each of the three tissues. A protein-protein interaction network inferred from previous work on Arabidopsis identified hub proteins whose transcripts show significant changes in expression. Glycolysis, the backbone of primary metabolism, appeared to be severely affected by the disorder, based on both transcriptomic and metabolomic results. Significantly less citric acid was observed consistently in puffed fruits. Gene set enrichment analysis suggested that glycolysis and carbohydrate metabolism were significantly altered in puffed samples in both albedo and flavedo. Expression of invertases and genes for sucrose export, amylose-starch and starch-maltose conversion was higher in puffed fruits. These changes may significantly alter source-sink communications. Genes associated with gibberellin and cytokinin signaling were downregulated in symptomatic albedo tissues, suggesting that these hormones play key roles in the disorder. Findings may be applied toward the development of early diagnostic methods based on host response genes and metabolites (i.e. citric acid), and toward therapeutics based on hormones.

Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia).

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.

Two-dimensional wavelet analysis based classification of gas chromatogram differential mobility spectrometry signals.

This study introduces two-dimensional (2-D) wavelet analysis to the classification of gas chromatogram differential mobility spectrometry (GC/DMS) data which are composed of retention time, compensation voltage, and corresponding intensities. One reported method to process such large data sets is to convert 2-D signals to 1-D signals by summing intensities either across retention time or compensation voltage, but it can lose important signal information in one data dimension. A 2-D wavelet analysis approach keeps the 2-D structure of original signals, while significantly reducing data size. We applied this feature extraction method to 2-D GC/DMS signals measured from control and disordered fruit and then employed two typical classification algorithms to testify the effects of the resultant features on chemical pattern recognition. Yielding a 93.3% accuracy of separating data from control and disordered fruit samples, 2-D wavelet analysis not only proves its feasibility to extract feature from original 2-D signals but also shows its superiority over the conventional feature extraction methods including converting 2-D to 1-D and selecting distinguishable pixels from training set. Furthermore, this process does not require coupling with specific pattern recognition methods, which may help ensure wide applications of this method to 2-D spectrometry data.

Superficial scald and bitter pit development in cold-stored transgenic apples suppressed for ethylene biosynthesis.

The plant hormone ethylene regulates climacteric fruit ripening and plays a major role in the development of superficial scald in apple fruits during cold storage. The effect of cold storage at 0 degrees C on development of superficial scald and bitter pit (BP) in transgenic Greensleeves (GS) apples suppressed for ethylene biosynthesis was investigated. Four apple lines were used: untransformed GS; line 68G, suppressed for 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO); and lines 103Yand 130Y, suppressed for ACC synthase (ACS). Fruits from the transformed lines 68G, 103Y, and 130Y produced very little ethylene during 3 months of cold storage at 0 degrees C and after subsequent transfer to 20 degrees C, whereas untransformed fruits produced significant ethylene during cold storage, which increased dramatically at 20 degrees C. Respiration, expressed as CO(2) production, was similar in all four apple lines. After 2 months at 0 degrees C, all apple lines showed some BP symptoms, but lines 68G and 103Y were more affected than untransformed GS or line 130Y. Both transformed and untransformed apples produced alpha-farnesene, but concentrations were lower in yellow fruit than in green fruit in all lines but 68G. Line 68G produced the most alpha-farnesene after 2 months at 0 degrees C, including both (E,E) alpha-farnesene and (Z,E) alpha-farnesene. Concentrations of (E,E) alpha-farnesene were 100 times greater than those of (Z,E) alpha-farnesene in all lines. After 4 months at 0 degrees C plus 1 week at 20 degrees C, untransformed GS apples exhibited the most superficial scald, whereas fruits from lines 68G and 103Y were less affected and line 130Y had no scald. Superficial scald severity was higher in green fruit than in yellow fruit in all affected lines. These lines also exhibited significant production of 6-methyl-5-hepten-2-one (MHO), a major oxidation product of (E,E) alpha-farnesene. Line 130Y neither exhibited superficial scald nor produced MHO. It is shown here that even transgenic apples suppressed for ethylene biosynthesis genes can produce alpha-farnesene, which in turn can oxidize to free radicals and MHO, leading to scald development.

Effect of gaseous ozone and hot water on microbial and sensory quality of cantaloupe and potential transference of Escherichia coli O157:H7 during cutting.

The effect of gaseous ozone and hot water, alone or in combination, on the sensory and microbial quality of cantaloupe melon was investigated. Escherichia coli O157:H7 transmission from the rind to edible melon flesh during cutting practices was also investigated. Four different treatments consisting of hot water (75 degrees C, 1min), gaseous ozone (10,000ppm, 30min), gaseous ozone supplied by carbon monoxide gas and the combination of hot water and gaseous ozone were evaluated. Sensory quality and growth evolution of aerobic mesophilic and psychrotrophic bacteria, coliforms and molds were studied. In general, hot water, gaseous ozone, and the combination of hot water and gaseous ozone were effective in reducing total microbial population. The combination of hot water and gaseous ozone was the most effective treatment to control microbial growth achieving 3.8, 5.1, 2.2 and 2.3log reductions for mesophilic and psychrotrophic bacteria, molds and coliforms, respectively. However no significant differences were observed between gaseous ozone and gaseous ozone supplied by with carbon monoxide gas. There was no evidence of damage in melons treated with hot water, ozone or their combination and they maintained initial texture and aroma. Therefore, the combination of hot water and gaseous ozone may be an efficient and promising treatment for controlling microbial growth and maintaining sensory quality of melons.

Viscoelastic properties of waxy and nonwaxy rice flours, their fat and protein-free starch, and the microstructure of their cooked kernels.

Physicochemistry and structural studies of two types of japonica rice, low amylose Calmochi-101 (CM101) and intermediate amylose M-202 (M202), were conducted to determine similarities and differences between the rices perhaps attributable to amylose content differences. The rheological behavior of the gelation and pasting processes of flours and starches was determined with high accuracy and precision using a controlled stress rheometer. Fat and protein, although minor constituents of milled rice, were shown to have significant effects on the physicochemical and pasting properties of starches and flours. Removal of protein and lipids with aqueous alkaline or detergent solutions caused lower pasting temperatures and higher overall viscosity in both starches, compared with their respective flours. There was less viscosity difference between M202 flour and its starch when isolated by enzymatic hydrolysis of protein. The protease did not reduce internally bound lipids, suggesting that fats help to determine pasting properties of rice flours and their respective starches. Structural integrity differences in individual granules of waxy and nonwaxy rice flours, starches, and whole raw, soaked, and cooked milled grain were revealed by fracture analysis and scanning electron microscopy. Calmochi 101 and M202 did not differ in weight-averaged molar mass (Mw) and root-mean-square radii (Rz) between flours and starches, as determined by high-performance size exclusion chromatography (HPSEC) and multiple-angle laser light scattering (MALLS) (Park, I.; Ibanez, A. M.; Shoemaker, C. F. Starch 2007, 59, 69-77).

Dissection of maize kernel composition and starch production by candidate gene association.

Cereal starch production forms the basis of subsistence for much of the world's human and domesticated animal populations. Starch concentration and composition in the maize (Zea mays ssp mays) kernel are complex traits controlled by many genes. In this study, an association approach was used to evaluate six maize candidate genes involved in kernel starch biosynthesis: amylose extender1 (ae1), brittle endosperm2 (bt2), shrunken1 (sh1), sh2, sugary1, and waxy1. Major kernel composition traits, such as protein, oil, and starch concentration, were assessed as well as important starch composition quality traits, including pasting properties and amylose levels. Overall, bt2, sh1, and sh2 showed significant associations for kernel composition traits, whereas ae1 and sh2 showed significant associations for starch pasting properties. ae1 and sh1 both associated with amylose levels. Additionally, haplotype analysis of sh2 suggested this gene is involved in starch viscosity properties and amylose content. Despite starch concentration being only moderately heritable for this particular panel of diverse maize inbreds, high resolution was achieved when evaluating these starch candidate genes, and diverse alleles for breeding and further molecular analysis were identified.