Roles of non-coding RNA in sugarcane-microbe interaction

Studies have highlighted the importance of non-coding RNA regulation in plant-microbe interaction. However, the roles of sugarcane microRNAs (miRNAs) in the regulation of disease responses have not been investigated. Firstly, we screened the sRNA transcriptome of sugarcane infected with Acidovorax avenae. Conserved and novel miRNAs were identified. Additionally, small interfering RNAs (siRNAs) were aligned to differentially expressed sequences from the sugarcane transcriptome. Interestingly, many siRNAs aligned to a transcript encoding a coppertransporter gene whose expression was induced in the presence of A. avenae, while the siRNAs were repressed in the presence of A. avenae. Moreover, a long intergenic non-coding RNA was identified as a potential target or decoy of miR408. To extend the bioinformatics analysis, we carried out independent inoculations and the expression patterns of six miRNAs were validated by quantitative reverse transcription-PCR (qRT-PCR). Among these miRNAs, miR408—a copper- microRNA—was downregulated. The cleavage of a putative miR408 target, a laccase, was confirmed by a modified 50RACE (rapid amplification of cDNA ends) assay. MiR408 was also downregulated in samples infected with other pathogens, but it was upregulated in the presence of a beneficial diazotrophic bacteria. Our results suggest that regulation by miR408 is important in sugarcane sensing whether microorganisms are either pathogenic or beneficial, triggering specific miRNA-mediated regulatory mechanisms accordingly

Salt stress induces changes in the proteomic profile of micropropagated sugarcane shoots

<div><p>Salt stress is one of the most common stresses in agricultural regions worldwide. In particular, sugarcane is affected by salt stress conditions, and no sugarcane cultivar presently show high productivity accompanied by a tolerance to salt stress. Proteomic analysis allows elucidation of the important pathways involved in responses to various abiotic stresses at the biochemical and molecular levels. Thus, this study aimed to analyse the proteomic effects of salt stress in micropropagated shoots of two sugarcane cultivars (CB38-22 and RB855536) using a label-free proteomic approach. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD006075. The RB855536 cultivar is more tolerant to salt stress than CB38-22. A quantitative label-free shotgun proteomic analysis identified 1172 non-redundant proteins, and 1160 of these were observed in both cultivars in the presence or absence of NaCl. Compared with CB38-22, the RB855536 cultivar showed a greater abundance of proteins involved in non-enzymatic antioxidant mechanisms, ion transport, and photosynthesis. Some proteins, such as calcium-dependent protein kinase, photosystem I, phospholipase D, and glyceraldehyde-3-phosphate dehydrogenase, were more abundant in the RB855536 cultivar under salt stress. Our results provide new insights into the response of sugarcane to salt stress, and the changes in the abundance of these proteins might be important for the acquisition of ionic and osmotic homeostasis during exposure to salt stress.</p></div

Relative expression profile of miRNA targets.

<p>Analysis of relative expression of miR159 target (GAMYB), miR164 target (NAC1), miR168 target (AGO1) and miR397 target (laccase) by qRT-PCR using samples of three sugarcane cultivar submitted to water depletion. In each case, control condition had relative expression equal 1 (dotted line). *represent significantly changing of miRNA expression between control and treatment samples (p-value <0.05).</p

Summary of results obtained after computational data mining of small RNA libraries.

a<p>Filtering for tRNA, rRNA, low-complexity sequence and trimming for “N” bases and 3′ adapters.</p>b<p>Conserved miRNA deposited at miRBase database identified by miRProf pipeline.</p

Distribution of leaf siRNA candidates aligned in cluster of siRNA.

<p>Each graph represents one leaf sRNA library. T = tolerant sugarcane cultivar, S = sensitive sugarcane cultivar. 0 h = control of stress, 24 h = time of water depletion treatment.</p

Validation of RNA-seq analysis by qRT-PCR using genes from different pathways.

<p>Two biological replicates were used. Gene names correspond to those listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166473#pone.0166473.s009" target="_blank">S9</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166473#pone.0166473.s010" target="_blank">S10</a> Tables. Relative expression by qRT-PCR. The bars represent the relative expression of three technical replicates (n = 3) and standard deviation (Green bars: replicate 1 and blue bars: replicate 2). The relative expression values above the dotted line are upregulated genes, whereas below line correspond to downregulated genes. GAPDH was used as a reference gene for normalization of gene-expression data. These 20 genes validated in replicates were grouped into four categories, <b>(A)</b> genes related to stress, <b>(B)</b> genes that coding to several pathways, <b>(C)</b> primary carbohydrate metabolism pathways genes and <b>(D)</b> genes encoding for PRRs. The values of the quantitative method ΔΔCt can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166473#pone.0166473.s010" target="_blank">S10 Table</a></p

Protein mapping of the CB38-22 and RB855536 cultivars under salt stress (180 mM NaCl) compared with their respective controls (without NaCl).

<p>A = CB-180/CB-0. B = RB-180/RB-0. All proteins with functions related to “response to biotic and abiotic stresses” are displayed on the maps. The log₂ values of the fold changes obtained for each cultivar under salt stress compared to their respective controls were used to determine the levels of protein abundance.</p

Sugarcane transcriptome analysis in response to infection caused by <i>Acidovorax avenae</i> subsp. <i>avenae</i>

<div><p>Sugarcane is an important tropical crop mainly cultivated to produce ethanol and sugar. Crop productivity is negatively affected by <i>Acidovorax avenae</i> subsp <i>avenae</i> (<i>Aaa</i>), which causes the red stripe disease. Little is known about the molecular mechanisms triggered in response to the infection. We have investigated the molecular mechanism activated in sugarcane using a RNA-seq approach. We have produced a <i>de novo</i> transcriptome assembly (TR7) from sugarcane RNA-seq libraries submitted to drought and infection with <i>Aaa</i>. Together, these libraries present 247 million of raw reads and resulted in 168,767 reference transcripts. Mapping in TR7 of reads obtained from infected libraries, revealed 798 differentially expressed transcripts, of which 723 were annotated, corresponding to 467 genes. GO and KEGG enrichment analysis showed that several metabolic pathways, such as code for proteins response to stress, metabolism of carbohydrates, processes of transcription and translation of proteins, amino acid metabolism and biosynthesis of secondary metabolites were significantly regulated in sugarcane. Differential analysis revealed that genes in the biosynthetic pathways of ET and JA PRRs, oxidative burst genes, NBS-LRR genes, cell wall fortification genes, SAR induced genes and pathogenesis-related genes (PR) were upregulated. In addition, 20 genes were validated by RT-qPCR. Together, these data contribute to a better understanding of the molecular mechanisms triggered by the <i>Aaa</i> in sugarcane and opens the opportunity for the development of molecular markers associated with disease tolerance in breeding programs.</p></div

22-nt miRNA and their target prediction.

a<p>start and end position of miRNA and target alignment.</p