Small RNA-mediated regulation of cross-kingdom gene expression in sugar beet genotypes resistant and susceptible to rhizomania

Abstract

Rhizomania in sugar beet causes significant yield and sucrose loss worldwide. The disease is caused by Beet necrotic yellow vein virus (BNYVV) and vectored by the plasmodiophorid, Polymyxa betae. Resistance to rhizomania in commercial cultivars is currently dependent upon the use of Rz1 and Rz2 resistant genes in sugar beet. We have developed an ethyl methanesulphonate mutant breeding line (KEMS12; PI672570) that is highly resistant to rhizomania. Using rhizomania-resistant (R) and susceptible (S) sugar beet breeding lines, natural infection and comprehensive RNA sequencing, we have identified the accumulation of a unique set of small non-coding RNAs (sncRNAs) derived from both the sugar beet plant and the BNYVV virus during active infection that may have possible regulatory roles in the resistance and/or susceptibility to rhizomania. Examples of target genes that are differentially expressed in the roots and leaves at early and late infection stages in sugar beet by plant-derived microRNAs (miRNAs) include Bevul.9G209500 (cytoplasm-related catalytic activity), Bevul.2G095700 (potassium transporter) and Bevul.9G160600 (zinc finger), which were up-regulated in the R line (vs. S). Viral-derived sncRNAs predominantly originated from RNA1 and RNA2 and targeted a subset of 69 sugar beet genes with overall expression that showed a strong negative correlation with higher sncRNA abundance. The results presented here for the first time demonstrate putative roles of sugar beet miRNAs in rhizomania resistance and BNYVV-derived sncRNAs and small peptides as potential pathogenicity factors.

Keywords: KEMS12; beet necrotic yellow vein virus; microRNA (miRNA); rhizomania; small non-coding RNA; sugar beet.

Fig. 1.. Disease phenotypes and viral load estimation in the rhizomania-resistant and -susceptible lines. (a) Plant stand in the rhizomania nursery at late infection stage. (b) Representative samples of uprooted plants at late infection stage. (c) Viral load in the roots. (d) Viral load in the leaves. Viral load was estimated as the ratio of total number of viral-derived transcripts/total number of sugar beet transcripts. Data are mean±SE of four biological replicates (eight individual plants/replicate). Rhizomania-resistant (KEMS12; 12), moderately susceptible (KEMS09; 09) and susceptible (KDH13; 13) sugar beet lines were used. ‘**’, P < 0.01; ‘*’, P < 0.05; and ‘+’, P<0.1 denote significant differences between lines for each treatment type. E_Rt, early root; L_Rt, late root; E_Lf, early leaf; L_Lf, late leaf.

Fig. 2.. Venn diagrams of DE miRNAs at different stages of rhizomania infection. (a) Early root (E_Rt). (b) Early leaf (E_Lf). (c) Late root (L_Rt). (d) Late leaf (L_Lf). Rhizomania-resistant (KEMS12; 12), moderately susceptible (KEMS09; 09) and susceptible (KDH13; 13) sugar beet lines were used. Data are mean±SE of four biological replicates (eight individual plants/replicate).

Fig. 3.. Heatmaps of the top 50 DE miRNAs (P < 0.0005) at different stages of rhizomania infection. (a) early root (E_Rt), (b) early leaf (E_Lf), (c) late root (L_Rt) and (d) late leaf (L_Lf) in the rhizomania-resistant (KEMS12; 12), moderately susceptible (KEMS09; 09) and susceptible (KDH13; 13) sugar beet lines. Data are from four biological replicates (eight individual plants/replicate).

Fig. 4.. KEGG enrichment of sugar beet target genes of DE sugar beet miRNAs at different stages of rhizomania infection. (a) Early root. (b) Late root. Data are mean±SE of four biological replicates (eight individual plants/replicate). DE miRNAs target sugar beet genes that alter their expressions.

Fig. 5.. Length distribution of sncRNAs across sugar beet root and leaf tissues at early and late infection stages. The sncRNAs were classified into three categories: all sncRNA (red), those putatively derived from BNYVV (blue) and BNYVV sncRNA alignment lengths to the EL10.2 sugar beet genome (green). Each category was sampled to 10,000 reads for comparison.

Fig. 6.. Abundance of BNYVV-derived sncRNAs in the beet roots and leaves of rhizomania-resistant (KEMS12; 12), moderately susceptible (KEMS09; 09) and susceptible (KDH13; 13) sugar beet lines at early and late infection stages. Data are mean±SE of four biological replicates (eight individual plants/replicate).

Fig. 7.. BNYVV sncRNA abundance and sugar beet mRNA expression. Scatterplot of sugar beet gene expression and count of sncRNAs.

Fig. 8.. Proposed model of possible interaction between BNYVV and the sugar beet host via miRNA and sncRNA. This generalized model highlights possible actors responsible for both infection and resistance to that infection in the case of up-regulated miRNAs in the resistant line (KEMS12). While this simplified diagram omits complexities that are likely to exist, it highlights the potential roles of miRNA and sncRNA in virus–host interaction.