RNAseq Transcriptional Profiling following Whip Development in Sugarcane Smut Disease

Abstract

Sugarcane smut disease is caused by the biotrophic fungus Sporisorium scitamineum. The disease is characterized by the development of a whip-like structure from the primary meristems, where billions of teliospores are produced. Sugarcane smut also causes tillering and low sucrose and high fiber contents, reducing cane productivity. We investigated the biological events contributing to disease symptoms in a smut intermediate-resistant sugarcane genotype by examining the transcriptional profiles (RNAseq) shortly after inoculating the plants and immediately after whip emission. The overall picture of disease progression suggests that premature transcriptional reprogramming of the shoot meristem functions continues until the emergence of the whip. The guidance of this altered pattern is potentially primarily related to auxin mobilization in addition to the involvement of other hormonal imbalances. The consequences associated with whip emission are the modulation of typical meristematic functions toward reproductive organ differentiation, requiring strong changes in carbon partitioning and energy production. These changes include the overexpression of genes coding for invertases and trehalose-6P synthase, as well as other enzymes from key metabolic pathways, such as from lignin biosynthesis. This is the first report describing changes in the transcriptional profiles following whip development, providing a hypothetical model and candidate genes to further study sugarcane smut disease progression.

Fig 1. Sugarcane smut.

(A) Scan electron microscopy of S. scitamineum hyphal growth in the sugarcane bud at 5 DAI (1, 2). Fungal sporogenesis and teliospore maturation in the base of the sugarcane whip at 200 DAI (3; 4). Bar = 10 μm. (B) (1) Sugarcane bud at 5 DAI. (2) Base of the whip at 200 DAI. Bar = 1 cm.

Fig 2. Differential expression gene analysis of 5-DAI and 200-DAI samples of the smut infected plants.

Venn diagrams show the number of genes detected as differentially expressed (DEGs) by the DESeq2 package [33] using two mapping software systems: 1) Bowtie2 and 2) BWA, as well as two sets of references: 1) sugarcane unigenes [29]and 2) the novel sugarcane transcripts assembled from sugarcane unigenes unmapped sequences using Trinity [32]. Intersections of the identified DEGs from the mapping results were considered for the following analysis.

Fig 3. Enrichment analysis of GO terms.

DEGs were submitted to enrichment analysis in BLAST2GO software, and a p-value ≤ 0.05 was used as the cut-off parameter. The gray bars represent the percentage of genes related to each selected GO term in the total set of sugarcane unigenes. The red bars represent the percentage of genes related to each selected GO term in the set of DEGs. The complete list of enriched GO terms in each set of DEGs can be found in S6 File.

Fig 4. Sugarcane DEGs related to meristem functions.

(A) Expression profile of genes related to meristem functions represented as values of a log2 fold change (inoculated/control). The heatmap was constructed using the R software package. Blue squares represent down-regulated genes, and red squares represent up-regulated ones. The statistical significance of expression regulation is presented in S5 and S6 Files. (B) Model of probable events related to whip development in sugarcane. Increase in VIN3 expression early in infected plants may release FT expression, which in turn positively regulates Apetala-1 (AP1) expression, turning the vegetative growth program to reproductive, via the autonomous/vernalization pathway. The black arrow represents up-regulation at 5 DAI, and the gray arrows represent up-regulation at 200 DAI.

Fig 5. RT-qPCR validation.

Sugarcane unigenes selected for RT-qPCR analysis of 5-DAI and 200-DAI samples: longifolia-like protein (comp200950_c0_seq1); auxin transporter (comp205699_c0_seq1); SAM (comp194455_c0_seq1); invertase (comp201528_c0_seq1); trehalose 6P synthase (comp204716_c0_seq1); aldolase (comp196354_c1_seq1); pyruvate decarboxylase (comp200606_c0_seq1) andperoxidase (comp187834_c0_seq1). The reactions were performed using a one-step GoTaq^® One-Step RT-qPCR System Kit (Promega) using a 7500 Fast Real-Time PCR System (Applied Biosystems). Statistical analysis was performed using REST^® software. “*” indicates genes differentially expressed in the RT-qPCR reactions (p-value < 0.05).

Fig 6. DEGs related to hormone biosynthesis and signalization.

(A) Heatmap constructed using the R software package and log2 fold change values (inoculated/control). The left column represents the regulation of each gene at 5 DAI, and the right column represents the regulation of each gene at 200 DAI. (B) Model concerning the contribution of the main plant hormones with up-regulated DEGs in sugarcane after whip emission.

Fig 7. Smutted sugarcane metabolism in the late moments of interaction.

Schematic representation of smutted sugarcane metabolism in the whip base (200 DAI). Red and blue arrows represent up- and down-regulated expression, respectively, and black arrows are unchanged expression.

Fig 8. Resistance gene analogs (RGAs) detected as DEGs in smut-infected sugarcane in early and late interaction.

The guide tree was obtained based on translated amino acid sequence similarity using CLUSTALW2. The heatmap represents the respective expression log2 fold change values. Protein structural features: signal peptide sequences predicted by SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/); transmembrane domains predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Other domains, motifs and active sites were predicted by InterProScan. CC, coiled coil; kinase (IPR011009); NB, nucleotide-binding (IPR002182); B, Bulb-type lectin domain (IPR001480); EGF, epidermal growth factor domain (IPR000742); PAN domain (IPR03609); LRR, leucine-rich-repeat (IPR001611; IPR003591);red pins represent kinase active sites, and gray arrows are P-loops of N-terminal NB-ARC proteins.