Allelic diversity of S‑RNase alleles in diploid potato species

S-ribonucleases (S-RNases) control the pistil specificity of the self-incompatibility (SI) response in the genus Solanum and several other members of the Solanaceae. The nucleotide sequences of S-RNases corresponding to a large number of S-alleles or S-haplotypes have been characterised. However, surprisingly few S-RNase sequences are available for potato species. The identification of new S-alleles in diploid potato species is desirable as these stocks are important sources of traits such as biotic and abiotic resistance. S-RNase sequences are reported here from three distinct diploid types of potato: cultivated Solanum tuberosum Group Phureja, S. tuberosum Group Stenotomum, and the wild species Solanum okadae. Partial S-RNase sequences were obtained from pistil RNA by RT-PCR or 3’RACE (Rapid Amplification of cDNA Ends) using a degenerate primer. Full length sequences were obtained for two alleles by 5’RACE. Database searches with these sequences, identified sixteen S-RNases in total, all of which are novel. The sequence analysis revealed all the expected features of functional S-RNases. Phylogenetic analysis with selected published S-RNase and S-like-RNase sequences from the Solanaceae revealed extensive trans-generic evolution of the S-RNases and a clear distinction from S-like-RNases. Pollination tests were used to confirm the self-incompatibility status and cross-compatibility relationships of the S. okadae accessions. All the S. okadae accessions were found to be self-incompatible as expected with crosses amongst them exhibiting both cross-compatibility and semi-compatibility consistent with the S-genotypes determined from the S-RNase sequence data. The progeny analysis of four semi-compatible crosses examined by allele-specific PCR provided further confirmation that these are functional S-RNases

Four plant defensins from an indigenous South African Brassicaceae species display divergent activities against two test pathogens despite high sequence similarity in the encoding genes

<p>Abstract</p> <p>Background</p> <p>Plant defensins are an important component of the innate defence system of plants where they form protective antimicrobial barriers between tissue types of plant organs as well as around seeds. These peptides also have other activities that are important for agricultural applications as well as the medical sector. Amongst the numerous plant peptides isolated from a variety of plant species, a significant number of promising defensins have been isolated from Brassicaceae species. Here we report on the isolation and characterization of four defensins from <it>Heliophila coronopifolia</it>, a native South African Brassicaceae species.</p> <p>Results</p> <p>Four defensin genes (<it>Hc-AFP1</it>-<it>4) </it>were isolated with a homology based PCR strategy. Analysis of the deduced amino acid sequences showed that the peptides were 72% similar and grouped closest to defensins isolated from other Brassicaceae species. The Hc-AFP1 and 3 peptides shared high homology (94%) and formed a unique grouping in the Brassicaceae defensins, whereas Hc-AFP2 and 4 formed a second homology grouping with defensins from <it>Arabidopsis </it>and <it>Raphanus</it>. Homology modelling showed that the few amino acids that differed between the four peptides had an effect on the surface properties of the defensins, specifically in the alpha-helix and the loop connecting the second and third beta-strands. These areas are implicated in determining differential activities of defensins. Comparing the activities after recombinant production of the peptides, Hc-AFP2 and 4 had IC₅₀values of 5-20 μg ml^-1against two test pathogens, whereas Hc-AFP1 and 3 were less active. The activity against <it>Botrytis cinerea </it>was associated with membrane permeabilization, hyper-branching, biomass reduction and even lytic activity. In contrast, only Hc-AFP2 and 4 caused membrane permeabilization and severe hyper-branching against the wilting pathogen <it>Fusarium solani</it>, while Hc-AFP1 and 3 had a mild morphogenetic effect on the fungus, without any indication of membrane activity. The peptides have a tissue-specific expression pattern since differential gene expression was observed in the native host. <it>Hc-AFP1 </it>and <it>3 </it>expressed in mature leaves, stems and flowers, whereas <it>Hc-AFP2 </it>and <it>4 </it>exclusively expressed in seedpods and seeds.</p> <p>Conclusions</p> <p>Two novel Brassicaceae defensin sequences were isolated amongst a group of four defensin encoding genes from the indigenous South African plant <it>H. coronopifolia</it>. All four peptides were active against two test pathogens, but displayed differential activities and modes of action. The expression patterns of the peptide encoding genes suggest a role in protecting either vegetative or reproductive structures in the native host against pathogen attack, or roles in unknown developmental and physiological processes in these tissues, as was shown with other defensins.</p

Harnessing the potential of ligninolytic enzymes for lignocellulosic biomass pretreatment

Abundant lignocellulosic biomass from various industries provides a great potential feedstock for the production of value-added products such as biofuel, animal feed, and paper pulping. However, low yield of sugar obtained from lignocellulosic hydrolysate is usually due to the presence of lignin that acts as a protective barrier for cellulose and thus restricts the accessibility of the enzyme to work on the cellulosic component. This review focuses on the significance of biological pretreatment specifically using ligninolytic enzymes as an alternative method apart from the conventional physical and chemical pretreatment. Different modes of biological pretreatment are discussed in this paper which is based on (i) fungal pretreatment where fungi mycelia colonise and directly attack the substrate by releasing ligninolytic enzymes and (ii) enzymatic pretreatment using ligninolytic enzymes to counter the drawbacks of fungal pretreatment. This review also discusses the important factors of biological pretreatment using ligninolytic enzymes such as nature of the lignocellulosic biomass, pH, temperature, presence of mediator, oxygen, and surfactant during the biodelignification process

Tratamento do feno de braquiária pelo fungo Pleurotus ostreatus Pretreatment effects on fiber degradation of brachiaria hay by Pleurotus ostreatus fungus

A inoculação de forragens com fungos lignocelulolíticos é uma opção para melhorar a qualidade destas sem adição de produtos químicos. O tratamento do substrato influencia a ação do fungo e a qualidade final do produto. Neste experimento, aplicaram-se quatro tratamentos (compostagem do feno inteiro, compostagem do feno picado, hidratação do feno em água fria e hidratação do feno em água quente) a um feno de Brachiaria decumbens. Aos tratamentos seguiu-se inoculação com o fungo Pleurotus ostreatus e incubação por 35 dias, sob temperatura controlada. Usou-se o delineamento inteiramente casualizado, com quatro repetições e medidas repetidas. Amostras foram colhidas semanalmente para acompanhar a degradação do substrato, mediante a análise química do feno. Observou-se aumento linear, com o decorrer do tempo, no teor de proteína bruta (PB) e na proporção de lignina na parede celular (LIG-FDN), e decréscimo linear nos valores de fibra em detergente neutro (FDN), celulose e hemicelulose. Não se observou efeito de tratamento no teor de FDA. Os tratamentos com compostagem apresentaram maiores valores de PB, lignina e LIG-FDN e menores de FDN e hemicelulose. Não se observou diferença entre os tratamentos com hidratação. O tratamento do feno de braquiária com o fungo propiciou degradação da fração fibrosa e aumento no teor de PB, com efeito mais intenso nos tratamentos que usaram compostagem. A ação do fungo foi mais efetiva sobre a hemicelulose que sobre os demais componentes da fibra.<br>The innoculation of forages with lignocellulolytic fungi is an option for improving quality without adding chemical products. Substrate quality influences fungal activity and endproduct quality. The effects of four treatments (composting of whole hay, composting of chopped hay, soaking in cool water and soaking in hot water) on a Brachiaria decumbens hay were evaluated. The treatments were followed by innoculation with Pleurotus ostreatus fungus and incubation over 35 days, under controlled temperature. A completely randomized design with four replicates and repeated measures was used. Weekly samples were taken to follow substrate degradation through chemical analysis of the hay. A linear increase over time was observed for crude protein (CP) and proportion of lignin in cell walls (LIG-NDF), whereas a linear decrease was observed for neutral detergent fiber (NDF), cellulose and hemicellulose contents. No treatment effect on ADF content was observed. The treatments based on composting showed higher CP, lignin and LIG-NDF contents and lower NDF and hemicellulose contents. No difference was observed between the treatments that used soaking. The biological treatment of Brachiaria hay caused degradation of the fibrous fraction and increased CP content, with stronger effect on those treatments that used composting. The fungus was more effective to increase hemicellulose content than the other fiber components