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.

Identification and characterization of core abscisic acid (ABA) signaling components and their gene expression profile in response to abiotic stresses in Setaria viridis.

Abscisic acid (ABA) is an essential phytohormone that regulates growth, development and adaptation of plants to environmental stresses. In Arabidopsis and other higher plants, ABA signal transduction involves three core components namely PYR/PYL/RCAR ABA receptors (PYLs), type 2C protein phosphatases (PP2Cs) and class III SNF-1-related protein kinase 2 (SnRK2s). In the present study, we reported the identification and characterization of the core ABA signaling components in Setaria viridis, an emerging model plant for cereals and feedstock crops presenting C4 metabolism, leading to the identification of eight PYL (SvPYL1 to 8), twelve PP2C (SvPP2C1 to 12) and eleven SnRK2 (SvSnRK2.1 through SvSnRK2.11) genes. In order to study the expression profiles of these genes, two different S. viridis accessions (A10.1 and Ast-1) were submitted to drought, salinity and cold stresses, in addition to application of exogenous ABA. Differential gene expression profiles were observed in each treatment and plant genotype, demonstrating variations of ABA stress responses within the same species. These differential responses to stresses were also assessed by physiological measurements such as photosynthesis, stomatal conductance and transpiration rate. This study allows a detailed analysis of gene expression of the core ABA signaling components in Setaria viridis submitted to different treatments and provides suitable targets for genetic engineering of C4 plants aiming tolerance to abiotic stresses.

Identification of candidate genes for drought tolerance in coffee by high-throughput sequencing in the shoot apex of different Coffea arabica cultivars.

BACKGROUND: Drought is a widespread limiting factor in coffee plants. It affects plant development, fruit production, bean development and consequently beverage quality. Genetic diversity for drought tolerance exists within the coffee genus. However, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. In this study, we compared the molecular responses to drought in two commercial cultivars (IAPAR59, drought-tolerant and Rubi, drought-susceptible) of Coffea arabica grown in the field under control (irrigation) and drought conditions using the pyrosequencing of RNA extracted from shoot apices and analysing the expression of 38 candidate genes. RESULTS: Pyrosequencing from shoot apices generated a total of 34.7 Mbp and 535,544 reads enabling the identification of 43,087 clusters (41,512 contigs and 1,575 singletons). These data included 17,719 clusters (16,238 contigs and 1,575 singletons) exclusively from 454 sequencing reads, along with 25,368 hybrid clusters assembled with 454 sequences. The comparison of DNA libraries identified new candidate genes (n = 20) presenting differential expression between IAPAR59 and Rubi and/or drought conditions. Their expression was monitored in plagiotropic buds, together with those of other (n = 18) candidates genes. Under drought conditions, up-regulated expression was observed in IAPAR59 but not in Rubi for CaSTK1 (protein kinase), CaSAMT1 (SAM-dependent methyltransferase), CaSLP1 (plant development) and CaMAS1 (ABA biosynthesis). Interestingly, the expression of lipid-transfer protein (nsLTP) genes was also highly up-regulated under drought conditions in IAPAR59. This may have been related to the thicker cuticle observed on the abaxial leaf surface in IAPAR59 compared to Rubi. CONCLUSIONS: The full transcriptome assembly of C. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. Using these data, candidate genes were selected and their differential expression profiles were confirmed by qPCR experiments in plagiotropic buds of IAPAR59 and Rubi under drought conditions. As regards the genes up-regulated under drought conditions, specifically in the drought-tolerant IAPAR59, several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as ABA and lipid metabolism, for example. The identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee.