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

The ten most frequently occurred protein domains in the sequence of sugarcane infected by <i>Aaa</i>.

<p>The ten most frequently occurred protein domains in the sequence of sugarcane infected by <i>Aaa</i>.</p

Workflow of analysis of the construction and analyses of the sugarcane reference TR7.

<p><b>(A)</b> The transcriptome assembly <i>De novo</i> was generated from sugarcane RNA libraries drought treated and libraries <i>Aaa</i> pathogen treated obtained from of Illumina. After was applied to quality filter to all raw reads the quality filter and next, were removed duplicate genome sequences from the dataset. The Velvet and Oases software was used for the <i>de novo</i> assembly of clean reads to generate the sugarcane reference transcriptome TR7 with 168,767 transcripts. <b>(B)</b> Differential expression analysis and annotated functional in TRAPID. About 14 millions of reads were mapping no TR7. Transcripts differentially expressed were selected by Fisher’s exact-test, p-value < 0.01 and transcripts that have the same expression on both replicas.</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

Schematic representation of the <i>Aaa</i> pathogen infection essay in sugarcane seedlings (<i>Saccharum</i> sp. SP70-1143 Cv) <i>in vitro</i> growth at 28°C.

<p><b>(A)</b> The of seedlings roots grown <i>in vitro</i> after 30 days were immersed in a suspension (10⁶ CFU ml-1) with <i>Aaa</i> and the other half used as a control seedlings were immersed in distilled water. Seedlings infected and controls were transferred to MS medium and kept for 7 days and after this period, whole plantlets were collected and immediately frozen in nitrogen. <b>(B)</b> Image of the control leaves without symptoms seedlings and <b>(C)</b> image of the early symptoms of red stripe disease caused by <i>Aaa in vitro</i> grown seedlings leaves.</p

Statistics of DETs annotated by TRAPID.

<p>Statistics of DETs annotated by TRAPID.</p

The top six up- and downregulated differentially expressed genes in sugarcane infected by <i>Aaa</i>.

<p>The top six up- and downregulated differentially expressed genes in sugarcane infected by <i>Aaa</i>.</p

Regulation of photosynthesis and carbohydrate metabolism in sugarcane infected with <i>Aaa</i>.

<p>Altered expression of genes in photosynthesis, glycolysis, metabolism starch, metabolism of raffinose, metabolism trehalose, sucrose metabolism, transport of sugar and mitochondrial respiratory chain. Red for downregulated and blue for upregulated genes. For additional details of the genes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166473#pone.0166473.s007" target="_blank">S7 Table</a>.</p

Histogram presentation of the GO enrichment analysis of sugarcane plantlets infected by <i>Aaa</i>.

<p>TRAPID system calculated GO enrichment based on the upregulated and downregulated dataset compared to a background (p-value 0.01). The x-axis indicates the percent of genes and the y-axis indicates the GO terms. GO analysis to <b>(A)</b> upregulated and <b>(B)</b> downregulated DEGs under biotic stress. For additional details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166473#pone.0166473.s004" target="_blank">S4 Table</a>.</p

Histogram presentation of the 8 KEGG metabolic pathways significantly enriched to DEGs of sugarcane plantlets infected by pathogen.

<p>The x-axis indicates the number of genes assigned to a specific pathway, the y-axis indicates the KEGG pathway. Enriched metabolic pathways to <b>(A)</b> upregulated and <b>(B)</b> downregulated DEGs. For additional details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166473#pone.0166473.s005" target="_blank">S5 Table</a>.</p