An Enzymatic and Proteomic Analysis of Panus lecomtei during Biodegradation of Gossypol in Cottonseed.

Cotton is an important plant-based protein. Cottonseed cake, a byproduct of the biodiesel industry, offers potential in animal supplementation, although the presence of the antinutritional sesquiterpenoid gossypol limits utilization. The macrofungus Panus lecomtei offers potential in detoxification of antinutritional factors. Through an enzymatic and proteomic analysis of P. lecomtei strain BRM044603, grown on crushed whole cottonseed contrasting in the presence of free gossypol (FG), this study investigated FG biodegradation over a 15-day cultivation period. Fungal growth reduced FG to levels at 100 µg/g, with a complex adaptive response observed, involving primary metabolism and activation of oxidative enzymes for metabolism of xenobiotics. Increasing activity of secreted laccases correlated with a reduction in FG, with enzyme fractions degrading synthetic gossypol to trace levels. A total of 143 and 49 differentially abundant proteins were observed across the two contrasting growth conditions after 6 and 12 days of cultivation, respectively, revealing a dynamic protein profile during FG degradation, initially related to constitutive metabolism, then later associated with responses to oxidative stress. The findings advance our understanding of the mechanisms involved in gossypol degradation and highlight the potential of P. lecomtei BRM044603 in cotton waste biotreatment, relevant for animal supplementation, sustainable resource utilization, and bioremediation.

Development of reference genes for RT-qPCR analysis of gene expression in Pleurotus pulmonarius for biotechnological applications.

Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed, inactivation of toxic phorbol esters present in the material is necessary. Pleurotus pulmonarius is a detoxifying agent for jatropha cake with additional potential as animal feed, edible mushroom and for enzyme production. For the characterization of fungal genes involved in phorbol ester degradation, together with other industrial applications, reverse transcription-quantitative PCR (RT-qPCR) is a tool that enables accurate quantification of gene expression. For this, reliable analysis requires reference genes for normalization of mRNA levels validated under conditions employed for target genes. The stability of potential reference genes ß-TUB, ACTIN, GAPDH, PHOS, EF1α, TRPHO, LAC, MNP3, MYP and VP were evaluated following growth of P. pulmonarius on toxic, non-toxic jatropha cake and a combined treatment, respectively. NormFinder and geNorm algorithms for expression stability analysis identified PHOS, EF1α and MNP3 as appropriate for normalizing gene expression. Reference gene combinations contrasting in ranking were compared following normalization of relative expression of the CHU_2040 gene, encoding an esterase enzyme potentially involved in phorbol ester degradation. The reference genes for P. pulmonarius will facilitate the elucidation of mechanisms involved in detoxification of phorbol esters as well as analysis of target genes for application in biorefinery models.

Ramularia leaf spot: PCR-based methods reveal widespread distribution of Ramulariopsis pseudoglycines and limited presence of R. gossypii in Brazil.

Whilst Brazil is the fourth largest cotton producer globally, incidence of ramularia leaf spot (RLS) has decreased yield. In 2017-18 and 2018-19, ca. 300 fungal samples were collected throughout Brazil. Hyphal tip cultures were obtained for amplification of the RNA polymerase II (RPB2), 28S rRNA, the ribosomal DNA internal transcribed spacers (ITS), actin (ACT), elongation factor (EF1-α) and histone H3 (HIS3) genomic regions. Additionally, sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained by nanopore sequencing and the EF1-α region was selected as a marker for rapid recognition of Ramulariopsis species. Clade assignments based on the concatenated-sequence tree were identical to those in tree generated by RPB2-sequences, as well as in an RPB2 haplotype network and an ISSR (TGTC)4 dendrogram, in identification with species-specific primers and based on morphological comparisons. Out of 267 examined isolates, 252 were identified as Ramulariopsis pseudoglycines, indicating this species as the most widespread causal agent of cotton RLS in the Brazilian growing regions. Species-specific primers developed in the study that target the EF1-α gene provide an opportunity for extensive RLS sampling worldwide to study the distribution of Ramulariopsis species. Such data will aid breeders and plant pathologists in cotton disease resistance development and fungicide resistance avoidance.

State-of-the-Art Molecular Plant Sciences in Brazil.

Brazil has a crucial role in global food security and biodiversity, boasting one of the largest agricultural areas and two globally vital biomes, the Amazon and the Atlantic Forest [...].

Characterization of microRNAs and Target Genes in Musa acuminata subsp. burmannicoides, var. Calcutta 4 during Interaction with Pseudocercospora musae.

Endogenous microRNAs (miRNAs) are small non-coding RNAs that perform post-transcriptional regulatory roles across diverse cellular processes, including defence responses to biotic stresses. Pseudocercospora musae, the causal agent of Sigatoka leaf spot disease in banana (Musa spp.), is an important fungal pathogen of the plant. Illumina HiSeq 2500 sequencing of small RNA libraries derived from leaf material in Musa acuminata subsp. burmannicoides, var. Calcutta 4 (resistant) after inoculation with fungal conidiospores and equivalent non-inoculated controls revealed 202 conserved miRNAs from 30 miR-families together with 24 predicted novel miRNAs. Conserved members included those from families miRNA156, miRNA166, miRNA171, miRNA396, miRNA167, miRNA172, miRNA160, miRNA164, miRNA168, miRNA159, miRNA169, miRNA393, miRNA535, miRNA482, miRNA2118, and miRNA397, all known to be involved in plant immune responses. Gene ontology (GO) analysis of gene targets indicated molecular activity terms related to defence responses that included nucleotide binding, oxidoreductase activity, and protein kinase activity. Biological process terms associated with defence included response to hormone and response to oxidative stress. DNA binding and transcription factor activity also indicated the involvement of miRNA target genes in the regulation of gene expression during defence responses. sRNA-seq expression data for miRNAs and RNAseq data for target genes were validated using stem-loop quantitative real-time PCR (qRT-PCR). For the 11 conserved miRNAs selected based on family abundance and known involvement in plant defence responses, the data revealed a frequent negative correlation of expression between miRNAs and target host genes. This examination provides novel information on miRNA-mediated host defence responses, applicable in genetic engineering for the control of Sigatoka leaf spot disease.

Transcriptome Profiling of the Resistance Response of Musa acuminata subsp. burmannicoides, var. Calcutta 4 to Pseudocercospora musae.

Banana (Musa spp.), which is one of the world's most popular and most traded fruits, is highly susceptible to pests and diseases. Pseudocercospora musae, responsible for Sigatoka leaf spot disease, is a principal fungal pathogen of Musa spp., resulting in serious economic damage to cultivars in the Cavendish subgroup. The aim of this study was to characterize genetic components of the early immune response to P. musae in Musa acuminata subsp. burmannicoides, var. Calcutta 4, a resistant wild diploid. Leaf RNA samples were extracted from Calcutta 4 three days after inoculation with fungal conidiospores, with paired-end sequencing conducted in inoculated and non-inoculated controls using lllumina HiSeq 4000 technology. Following mapping to the reference M. acuminata ssp. malaccensis var. Pahang genome, differentially expressed genes (DEGs) were identified and expression representation analyzed on the basis of gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes orthology and MapMan pathway analysis. Sequence data mapped to 29,757 gene transcript models in the reference Musa genome. A total of 1073 DEGs were identified in pathogen-inoculated cDNA libraries, in comparison to non-inoculated controls, with 32% overexpressed. GO enrichment analysis revealed common assignment to terms that included chitin binding, chitinase activity, pattern binding, oxidoreductase activity and transcription factor (TF) activity. Allocation to KEGG pathways revealed DEGs associated with environmental information processing, signaling, biosynthesis of secondary metabolites, and metabolism of terpenoids and polyketides. With 144 up-regulated DEGs potentially involved in biotic stress response pathways, including genes involved in cell wall reinforcement, PTI responses, TF regulation, phytohormone signaling and secondary metabolism, data demonstrated diverse early-stage defense responses to P. musae. With increased understanding of the defense responses occurring during the incompatible interaction in resistant Calcutta 4, these data are appropriate for the development of effective disease management approaches based on genetic improvement through introgression of candidate genes in superior cultivars.

Stable reference genes for RT-qPCR analysis of gene expression in the Musa acuminata-Pseudocercospora musae interaction.

Leaf pathogens are limiting factors in banana (Musa spp.) production, with Pseudocercospora spp. responsible for the important Sigatoka disease complex. In order to investigate cellular processes and genes involved in host defence responses, quantitative real-time PCR (RT-qPCR) is an analytical technique for gene expression quantification. Reliable RT-qPCR data, however, requires that reference genes for normalization of mRNA levels in samples are validated under the conditions employed for expression analysis of target genes. We evaluated the stability of potential reference genes ACT1, α-TUB, UBQ1, UBQ2, GAPDH, EF1α, APT and RAN. Total RNA was extracted from leaf tissues of Musa acuminata genotypes Calcutta 4 (resistant) and Cavendish Grande Naine (susceptible), both subjected to P. musae infection. Expression stability was determined with NormFinder, BestKeeper, geNorm and RefFinder algorithms. UBQ2 and RAN were the most stable across all M. acuminata samples, whereas when considering inoculated and non-inoculated leaf samples, APT and UBQ2 were appropriate for normalization in Calcutta 4, with RAN and α-TUB most stable in Cavendish Grande Naine. This first study of reference genes for relative quantification of target gene expression in the M. acuminata-P. musae interaction will enable reliable analysis of gene expression in this pathosystem, benefiting elucidation of disease resistance mechanisms.

Phylogenetic relationships of Phaeochorellaparinarii and recognition of a new family, Phaeochorellaceae (Diaporthales).

Species of Phaeochorella are biotrophic leaf parasites with a tropical distribution, traditionally accepted in the family Phyllachoraceae, Phyllachorales in classifications based on morphological characters. Phylogenetic evidence presented here resolves the relationship of Phaeochorella within the Sordariomycetes, based on a multilocus analysis of partial nuc rDNA large subunit (28S) and internal transcribed spacers (ITS1-5.8S-ITS2 = ITS), the DNA-directed RNA polymerase II second largest subunit (RPB2), and the translation elongation factor 1-α (TEF1-α) gene. Phylogenetic analyses indicate that Phaeochorella belongs to the Diaporthales rather than the Phyllachorales. Phaeochorella parinarii, the type species of the genus, present on native hosts from the Brazilian Cerrado, forms a unique clade with a species of Phaeoappendicospora with high support. Thus, a new family, Phaeochorellaceae, Diaporthales, including both genera, is herein proposed. With the exception of P. parinarii and P. zonata, all other species in Phaeochorella (P. artocarpi, P. ciliata, P. machaerii) were excluded from the genus.

Analysis of the Transcriptome in Aspergillus tamarii During Enzymatic Degradation of Sugarcane Bagasse.

The production of bioethanol from non-food agricultural residues represents an alternative energy source to fossil fuels for incorporation into the world's economy. Within the context of bioconversion of plant biomass into renewable energy using improved enzymatic cocktails, Illumina RNA-seq transcriptome profiling was conducted on a strain of Aspergillus tamarii, efficient in biomass polysaccharide degradation, in order to identify genes encoding proteins involved in plant biomass saccharification. Enzyme production and gene expression was compared following growth in liquid and semi-solid culture with steam-exploded sugarcane bagasse (SB) (1% w/v) and glucose (1% w/v) employed as contrasting sole carbon sources. Enzyme production following growth in liquid minimum medium supplemented with SB resulted in 0.626 and 0.711 UI.mL-1 xylanases after 24 and 48 h incubation, respectively. Transcriptome profiling revealed expression of over 7120 genes, with groups of genes modulated according to solid or semi-solid culture, as well as according to carbon source. Gene ontology analysis of genes expressed following SB hydrolysis revealed enrichment in xyloglucan metabolic process and xylan, pectin and glucan catabolic process, indicating up-regulation of genes involved in xylanase secretion. According to carbohydrate-active enzyme (CAZy) classification, 209 CAZyme-encoding genes were identified with significant differential expression on liquid or semi-solid SB, in comparison to equivalent growth on glucose as carbon source. Up-regulated CAZyme-encoding genes related to cellulases (CelA, CelB, CelC, CelD) and hemicellulases (XynG1, XynG2, XynF1, XylA, AxeA, arabinofuranosidase) showed up to a 10-fold log2FoldChange in expression levels. Five genes from the AA9 (GH61) family, related to lytic polysaccharide monooxygenase (LPMO), were also identified with significant expression up-regulation. The transcription factor gene XlnR, involved in induction of hemicellulases, showed up-regulation on liquid and semi-solid SB culture. Similarly, the gene ClrA, responsible for regulation of cellulases, showed increased expression on liquid SB culture. Over 150 potential transporter genes were also identified with increased expression on liquid and semi-solid SB culture. This first comprehensive analysis of the transcriptome of A. tamarii contributes to our understanding of genes and regulatory systems involved in cellulose and hemicellulose degradation in this fungus, offering potential for application in improved enzymatic cocktail development for plant biomass degradation in biorefinery applications.

Taxonomy, phylogeny, and divergence time estimation for Apiosphaeria guaranitica, a Neotropical parasite on bignoniaceous hosts.

Apiosphaeria guaranitica, the causal agent of brown crust disease of several bignoniaceous hosts, among them Handroanthus and Tabebuia species, has been traditionally placed in Phyllachoraceae, based exclusively on morphological studies, without supporting molecular evidence. Here, we provide molecular data for the link between sexual and asexual states of the fungus and elucidate the phylogeny of A. guaranitica. The multilocus phylogenetic analyses employed sequences from the 18S subunit (18S), 28S subunit (28S), and nuclear internal transcribed spacers (ITS1-5.8S-ITS2 = ITS) of the nuc rDNA, second-largest subunit of RNA polymerase II (RPB2), and translation elongation factor 1-α (TEF1) genetic loci. Estimates of the divergence time of this lineage were supported by fossil calibration (FC) and secondary calibration (SC) strategies. Our results indicate a natural placement of Apiosphaeria within Diaporthaceae (Diaporthales), where it represents an ancient lineage of the crown group of Diaporthaceae, diverging during the late Paleocene at 61.15 (FC) and 60.63 (SC) million years ago. This divergence time estimate within Diaporthales is based on Spataporthe taylori, a diaporthaceous fossil.

Development of an RP-UHPLC-PDA method for quantification of free gossypol in cottonseed cake and fungal-treated cottonseed cake.

Cottonseed cake biomass, which is a residue of oil extraction, is potentially appropriate for use as animal feed, given the high mineral, fibre and protein content. The presence of free gossypol, however, a toxic pigment in the glands of the cotton plant, limits use of this biomass for monogastric livestock. A promising method to detoxify cottonseed cake relies on fermentation by fungi, which can eliminate up to 100% of gossypol. In order to quantify trace levels of free gossypol in different cotton materials, including cottonseed cake treated with macrofungi, a simple and rapid chromatographic detection method was developed and validated. Under optimized conditions, extraction was performed using 70% acetone. The extract was then analysed by Ultra High-Performance Liquid Chromatography (UHPLC), with gradient elution on a C18 reverse phase column KINETEX® (100 x 2.10 mm, 2.6 µm). Methanol-0.1% TFA aqueous solution was employed as mobile phase and PDA detection conducted at 254 nm. The optimized method was validated by analysis of specificity, linearity and range, limit of detection, limit of quantification, precision and accuracy. Detection and quantification limits were observed at 0.2 and 0.5 µg/mL, respectively. With good reproducibility, with precision (RSD)<10% and recovery greater than 94%, the developed assay was appropriate for quantification of low quantities of free gossypol. The validated method was successfully applied to determine trace levels of free gossypol cottonseed treated with a macrofungus.

Identification of multienzymatic complexes in the Clonostachys byssicola secretomes produced in response to different lignocellulosic carbon sources.

Multienzymatic complexes with plant lignocellulose-degrading activities have recently been identified in filamentous fungi secretomes. Such complexes have potential biotechnological applications in the degradation of agro-industrial residues. Fungal species from the Clonostachys genus have been intensively investigated as biocontrol agents; however so far their use as producers of lignocellulose-degrading enzymes has not been extensively explored. Secretomes of Clonostachys byssicola following growth on different carbon sources (passion fruit peel, soybean hulls, cotton gin trash, banana stalk, sugarcane bagasse, orange peel, and a composition of soybean hulls: cotton gin trash:orange peel) were subjected to enzymatic assays. Remarkable differences were observed among the samples, especially regarding levels of mannanase and pectinase activities. Secretomes were then subjected to Blue Native PAGE in order to resolve putative protein complexes which subsequently had their composition revealed by trypsin digestion followed by LC-MS/MS analysis. The protein bands (named I, II, III and IV) were shown to be composed by holocellulolytic enzymes, mainly cellulases and xylanases as well as proteins involved in biocontrol processes, such as chitinases and proteases. The high diversity of proteins found in these multicatalytic assemblies confirms C. byssicola as a novel source of plant biomass-degrading enzymes.

Plant immunity: unravelling the complexity of plant responses to biotic stresses.

Background: Plants are constantly exposed to evolving pathogens and pests, with crop losses representing a considerable threat to global food security. As pathogen evolution can overcome disease resistance that is conferred by individual plant resistance genes, an enhanced understanding of the plant immune system is necessary for the long-term development of effective disease management strategies. Current research is rapidly advancing our understanding of the plant innate immune system, with this multidisciplinary subject area reflected in the content of the 18 papers in this Special Issue. Scope: Advances in specific areas of plant innate immunity are highlighted in this issue, with focus on molecular interactions occurring between plant hosts and viruses, bacteria, phytoplasmas, oomycetes, fungi, nematodes and insect pests. We provide a focus on research across multiple areas related to pathogen sensing and plant immune response. Topics covered are categorized as follows: binding proteins in plant immunity; cytokinin phytohormones in plant growth and immunity; plant-virus interactions; plant-phytoplasma interactions; plant-fungus interactions; plant-nematode interactions; plant immunity in Citrus; plant peptides and volatiles; and assimilate dynamics in source/sink metabolism. Conclusions: Although knowledge of the plant immune system remains incomplete, the considerable ongoing scientific progress into pathogen sensing and plant immune response mechanisms suggests far reaching implications for the development of durable disease resistance against pathogens and pests.

Gene expression analysis in Musa acuminata during compatible interactions with Meloidogyne incognita.

Background and Aims: Endoparasitic root-knot nematodes (RKNs) ( Meloidogyne spp.) cause considerable losses in banana ( Musa spp.), with Meloidogyne incognita a predominant species in Cavendish sub-group bananas. This study investigates the root transcriptome in Musa acuminata genotypes 4297-06 (AA) and Cavendish Grande Naine (CAV; AAA) during early compatible interactions with M. incognita . Methods: Roots were analysed by brightfield light microscopy over a 35 d period to examine nematode penetration and morphological cell transformation. RNA samples were extracted 3, 7 and 10 days after inoculation (DAI) with nematode J2 juveniles, and cDNA libraries were sequenced using lllumina HiSeq technology. Sequences were mapped to the M. acuminata ssp. malaccensis var. Pahang genome sequence, differentially expressed genes (DEGs) identified and transcript representation determined by gene set enrichment and pathway mapping. Key Results: Microscopic analysis revealed a life cycle of M. incognita completing in 24 d in CAV and 27 d in 4279-06. Comparable numbers of DEGs were up- and downregulated in each genotype, with potential involvement of many in early host defence responses involving reactive oxygen species and jasmonate/ethylene signalling. DEGs revealed concomitant auxin metabolism and cell wall modification processes likely to be involved in giant cell formation. Notable transcripts related to host defence included those coding for leucine-rich repeat receptor-like serine/threonine-protein kinases, peroxidases, thaumatin-like pathogenesis-related proteins, and DREB, ERF, MYB, NAC and WRKY transcription factors. Transcripts related to giant cell development included indole acetic acid-amido synthetase GH3.8 genes, involved in auxin metabolism, as well as genes encoding expansins and hydrolases, involved in cell wall modification. Conclusions: Expression analysis in M. acuminata during compatible interactions with RKNs provides insights into genes modulated during infection and giant cell formation. Increased understanding of both defence responses to limit parasitism during compatible interactions and effector-targeted host genes in this complex interaction will facilitate the development of genetic improvement measures for RKNs.

GH11 xylanase from Emericella nidulans with low sensitivity to inhibition by ethanol and lignocellulose-derived phenolic compounds.

An endo-ß-1,4-xylanase (X22) was purified from crude extract of Emericella nidulans when cultivated on submerged fermentation using sugarcane bagasse as the carbon source. The purified protein was identified by mass spectrometry and was most active at pH and temperature intervals of 5.0-6.5 and 50-60°C, respectively. The enzyme showed half-lives of 40, 10 and 7 min at 28, 50 and 55°C, respectively, and pH 5.0. Apparent Km and Vmax values on soluble oat spelt xylan were 3.39 mg/mL and 230.8 IU/mg, respectively, while Kcat and Kcat/Km were 84.6 s(-1) and 25.0 s(-1) mg(-1) mL. Incubation with phenolic compounds showed that tannic acid and cinnamic acid had an inhibitory effect on X22 but no time-dependent deactivation. On the other hand, ferulic acid, 4-hydroxybenzoic acid, vanillin and p-coumaric acid did not show any inhibitory effect on X22 activity, although they changed X22 apparent kinetic parameters. Ethanol remarkably increased enzyme thermostability and apparent Vmax and Kcat values, even though the affinity and catalytic efficiency for xylan were lowered.

Identification of mycoparasitism-related genes against the phytopathogen Sclerotinia sclerotiorum through transcriptome and expression profile analysis in Trichoderma harzianum.

BACKGROUND: The species of T. harzianum are well known for their biocontrol activity against plant pathogens. However, few studies have been conducted to further our understanding of its role as a biological control agent against S. sclerotiorum, a pathogen involved in several crop diseases around the world. In this study, we have used RNA-seq and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum gene expression during growth on cell wall of S. sclerotiorum (SSCW) or glucose. RT-qPCR was also used to examine genes potentially involved in biocontrol, during confrontation between T. harzianum and S. sclerotiorum. RESULTS: Data obtained from six RNA-seq libraries were aligned onto the T. harzianum CBS 226.95 reference genome and compared after annotation using the Blast2GO suite. A total of 297 differentially expressed genes were found in mycelia grown for 12, 24 and 36 h under the two different conditions: supplemented with glucose or SSCW. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on SSCW or glucose. We identified various genes of biotechnological value encoding proteins with functions such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. To validate the expression profile, RT-qPCR was performed using 20 randomly chosen genes. RT-qPCR expression profiles were in complete agreement with the RNA-Seq data for 17 of the genes evaluated. The other three showed differences at one or two growth times. During the confrontation assay, some genes were up-regulated during and after contact, as shown in the presence of SSCW which is commonly used as a model to mimic this interaction. CONCLUSIONS: The present study is the first initiative to use RNA-seq for identification of differentially expressed genes in T. harzianum strain TR274, in response to the phytopathogenic fungus S. sclerotiorum. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against S.sclerotiorum. The RNA-seq data presented will facilitate improvement of the annotation of gene models in the draft T. harzianum genome and provide important information regarding the transcriptome during this interaction.