Sugarcane mosaic virus mediated changes in cytosine methylation pattern and differentially transcribed fragments in resistance-contrasting sugarcane genotypes

Abstract

Sugarcane mosaic virus (SCMV) is the causal agent of sugarcane mosaic disease (SMD) in Brazil; it is mainly controlled by using resistant cultivars. Studies on the changes in sugarcane transcriptome provided the first insights about the molecular basis underlying the genetic resistance to SMD; nonetheless, epigenetic modifications such as cytosine methylation is also informative, considering its roles in gene expression regulation. In our previous study, differentially transcribed fragments (DTFs) were obtained using cDNA-amplified fragment length polymorphism by comparing mock- and SCMV-inoculated plants from two sugarcane cultivars with contrasting responses to SMD. In this study, the identification of unexplored DTFs was continued while the same leaf samples were used to evaluate SCMV-mediated changes in the cytosine methylation pattern by using methylation-sensitive amplification polymorphism. This analysis revealed minor changes in cytosine methylation in response to SCMV infection, but distinct changes between the cultivars with contrasting responses to SMD, with higher hypomethylation events 24 and 72 h post-inoculation in the resistant cultivar. The differentially methylated fragments (DMFs) aligned with transcripts, putative promoters, and genomic regions, with a preponderant distribution within CpG islands. The transcripts found were associated with plant immunity and other stress responses, epigenetic changes, and transposable elements. The DTFs aligned with transcripts assigned to stress responses, epigenetic changes, photosynthesis, lipid transport, and oxidoreductases, in which the transcriptional start site is located in proximity with CpG islands and tandem repeats. Real-time quantitative polymerase chain reaction results revealed significant upregulation in the resistant cultivar of aspartyl protease and VQ protein, respectively, selected from DMF and DTF alignments, suggesting their roles in genetic resistance to SMD and supporting the influence of cytosine methylation in gene expression. Thus, we identified new candidate genes for further validation and showed that the changes in cytosine methylation may regulate important mechanisms underlying the genetic resistance to SMD.

Fig 1. Principal coordinate analysis (PCoA) for A: methylation-susceptible loci (MSL) and B: nonmethylated loci (NML), representing epigenetic and genetic differences among groups, respectively.

Fig 2

a) Box-plot representing the distribution of the 72 raw Ct values for each gene. The whiskers denote the highest and lowest Ct values, whereas the lower and upper boundaries of the box (interquartile) represent the 25^th and 75^th percentile, respectively. The mean values of each gene are represented by the line within the boxes. b) Average relative normalized expression of the three transcripts aligned to differentially transcribed fragments (DTFs), i.e., Sh_005D21_g0060, Sh_250G13_g000040, and SCQGST1032C04.g, and two transcripts aligned to differentially methylated fragments (DMFs), i.e., Sh_206E04_g000020 and SP803280_c104096_g2_i1, selected based on their assigned annotations from Uniprot. Results represent the fold change in comparison with mock-inoculated samples, normalized to the transcript abundance of ubiquitin-conjugating enzyme 18 (UBC18) and uridylate kinase (UK). Error bars indicate standard error of the mean (n = 3 biological replicates from a single experiment). Asterisks (*p< 0.05; **p< 0.01) indicate statistically significant differences of the mean values determined using Student’s t-test.