Cytosine Methylation in Genomic DNA and Characterization of DNA Methylases and Demethylases and Their Expression Profiles in Viroid-Infected Hop Plants (Humulus lupulus Var. ‘Celeia’)

Abstract

Abiotic and biotic stresses can lead to changes in host DNA methylation, which in plants is also mediated by an RNA-directed DNA methylation mechanism. Infections with viroids have been shown to affect DNA methylation dynamics in different plant hosts. The aim of our research was to determine the content of 5-methylcytosine (5-mC) in genomic DNA at the whole genome level of hop plants (Humulus lupulus Var. ‘Celeia’) infected with different viroids and their combinations and to analyse the expression of the selected genes to improve our understanding of DNA methylation dynamics in plant-viroid systems. The adapted HPLC-UV method used proved to be suitable for this purpose, and thus we were able to estimate for the first time that the cytosine methylation level in viroid-free hop plants was 26.7%. Interestingly, the observed 5-mC level was the lowest in hop plants infected simultaneously with CBCVd, HLVd and HSVd (23.7%), whereas the highest level was observed in plants infected with HLVd (31.4%). In addition, we identified three DNA methylases and one DNA demethylase gene in the hop’s draft genome. The RT-qPCR revealed upregulation of all newly identified genes in hop plants infected with all three viroids, while no altered expression was observed in any of the other hop plants tested, except for CBCVd-infected hop plants, in which one DNA methylase was also upregulated.

Integrative time‐scale and multi‐omics analysis of host responses to viroid infection

“…Consequently, epigenetic epidemiology has emerged as a promising area for future research on infectious diseases (Gómez‐Díaz et al, 2012). Recent studies have evidenced dynamic changes in host DNA methylation patterns upon viroid infection in both somatic (Castellano, Pallas, et al, 2016; Lv et al, 2016; Martinez et al, 2014; Sečnik et al, 2022; Torchetti et al, 2016) and reproductive (Castellano, Martinez, et al, 2016) tissues. The changes of the host‐genome epigenetic regulation have been connected (in infected cucumber plants) with viroid recruiting and functionally subverting the host HISTONE DEACETYLASE 6 (HDA6) (Castellano, Pallas, et al, 2016).…”

PSTVd infection in Nicotiana benthamiana plants has a minor yet detectable effect on CG methylation

“…Nevertheless, how viroids are using this mechanism remains unclear. PSTVd and HLVd have been suggested to induce hypermethylation in potato and hop plants respectively (Lv et al, 2016;Secňik et al, 2022), whereas HSVd induces hypomethylation in promoters and TE of cucumber and N. benthamiana plants (Martinez et al, 2014;Castellano et al, 2015;Castellano et al, 2016a;Castellano et al, 2016b). In addition, upon co-infection of PSTVd and a geminivirus, hypermethylation of viral DNA was observed while during a triple co-infection in hop plants with CBCVd, HLVd and HSVd, genomic hypomethylation was reported (Torchetti et al, 2016;Secňik et al, 2022).…”

“…PSTVd and HLVd have been suggested to induce hypermethylation in potato and hop plants respectively (Lv et al, 2016;Secňik et al, 2022), whereas HSVd induces hypomethylation in promoters and TE of cucumber and N. benthamiana plants (Martinez et al, 2014;Castellano et al, 2015;Castellano et al, 2016a;Castellano et al, 2016b). In addition, upon co-infection of PSTVd and a geminivirus, hypermethylation of viral DNA was observed while during a triple co-infection in hop plants with CBCVd, HLVd and HSVd, genomic hypomethylation was reported (Torchetti et al, 2016;Secňik et al, 2022). Since viroids do not encode for proteins, any effect on DNA methylation could result from the activity of DCL produced vd-siRNAs that bind methylation-related AGO proteins and therefore potentially activate RdDM pathways (Dadami et al, 2013;Minoia et al, 2014;Katsarou et al, 2016a;Katsarou et al, 2022).…”

“…Furthermore, specific endogenous promoter regions of PSTVd-infected potato plants were shown to be hypermethylated, possibly with the implication of a known viroid partner, protein VirP1 (Kalantidis et al, 2007;Lv et al, 2016). In a recent study investigating genomic methylation levels in hop plants infected with various viroids, only hop latent viroid (HLVd) showed hypermethylation (Secňik et al, 2022). On the contrary, in hop stunt viroid (HSVd) infections of either cucumber or N. benthamiana plants promoter regions of ribosomal genes and TEs were found hypomethylated through a direct interaction with HDA6 (HISTONE DEACETYLASE 6) protein, a known RdDM partner (Martinez et al, 2014;Castellano et al, 2015;Castellano et al, 2016a;Castellano et al, 2016b).…”

“…On the contrary, in hop stunt viroid (HSVd) infections of either cucumber or N. benthamiana plants promoter regions of ribosomal genes and TEs were found hypomethylated through a direct interaction with HDA6 (HISTONE DEACETYLASE 6) protein, a known RdDM partner (Martinez et al, 2014;Castellano et al, 2015;Castellano et al, 2016a;Castellano et al, 2016b). In addition, during coinfections of PSTVd with a DNA virus (Geminiviridae) in tomato plants, hypermethylation of viral DNA was observed, whereas in coinfection of three viroids [citrus bark cracking viroid (CBCVd), HLVd and HSVd] a global genome hypomethylation was reported (Torchetti et al, 2016;Secňik et al, 2022). All these studies show the degree of contradictory results concerning DNA methylation effect in viroid infections, and the need for supplementary studies to elucidate the phenomenon.…”