Tomato yellow leaf curl virus resistance by Ty-1 involves increased cytosine methylation of viral genomes and is compromised by cucumber mosaic virus infection

Abstract

Tomato yellow leaf curl virus (TYLCV) and related begomoviruses are a major threat to tomato production worldwide and, to protect against these viruses, resistance genes from different wild tomato species are introgressed. Recently, the Ty-1 resistance gene was identified, shown to code for an RNA-dependent RNA polymerase and to be allelic with Ty-3. Here we show that upon TYLCV challenging of resistant lines carrying Ty-1 or Ty-3, low virus titers were detected concomitant with the production of relatively high levels of siRNAs whereas, in contrast, susceptible tomato Moneymaker (MM) revealed higher virus titers but lower amounts of siRNAs. Comparative analysis of the spatial genomic siRNA distribution showed a consistent and subtle enrichment for siRNAs derived from the V1 and C3 genes in Ty-1 and Ty-3. In plants containing Ty-2 resistance the virus was hardly detectable, but the siRNA profile resembled the one observed in TYLCV-challenged susceptible tomato (MM). Furthermore, a relative hypermethylation of the TYLCV V1 promoter region was observed in genomic DNA collected from Ty-1 compared with that from (MM). The resistance conferred by Ty-1 was also effective against the bipartite tomato severe rugose begomovirus, where a similar genome hypermethylation of the V1 promoter region was discerned. However, a mixed infection of TYLCV with cucumber mosaic virus compromised the resistance. The results indicate that Ty-1 confers resistance to geminiviruses by increasing cytosine methylation of viral genomes, suggestive of enhanced transcriptional gene silencing. The mechanism of resistance and its durability toward geminiviruses under natural field conditions is discussed.

Keywords: RNAi; Solanaceae.

Fig. 1.

TYLCV-derived siRNA in plants from different tomato lines. (A) Schematic genome organization of TYLCV harboring six ORFs: V2, precoat protein; V1, coat protein; C3, replicator enhancer protein; C2, transcriptional activator protein; C1, replicator initiator protein; and C4, symptom and movement determinant protein. IR, intergenic region. (B) Relative abundance (no internal control sample) of TYLCV-derived siRNAs determined by Southern blot. siRNAs isolated from TYLCV-infected plants of the susceptible cultivar Moneymaker and resistant Ty-1, Ty-2, and Ty-3 lines were radiolabeled and hybridized to six PCR fragments (minor rearrangements in blots for MM and Ty-1 to present in clockwise section order) representing the entire TYLCV genome (the corresponding sections of the fragments are indicated by black bars outside the genome circle in A). As negative controls, siRNAs were isolated from mock inoculated MM and Ty-1 plants and used as a probe (Upper Right). siRNA concentrations were quantified in CNT per mm², defined as the sum of all intensities.

Fig. 2.

Quantification of TYLCV-derived siRNA levels relative to an internal GFP-siRNA control. siRNAs isolated from TYLCV-infected susceptible MM and resistant Ty-1 plants were radiolabeled and hybridized to PCR fragments representing the entire TYLCV genome (fragment sections shown as V1, V2, and C1–C4 are indicated in the genome scheme in Fig. 1A). Bars indicate SDs for the relative siRNA concentration based on three replicates.

Fig. 3.

DNA methylation analysis of TYLCV isolated from MM and Ty-1 plants infected with TYLCV. Schematic overview of the multiple sequence alignment of 20 cloned PCR products per tomato plant obtained from bisulfite-treated DNA genome templates. Black bars indicate the positions of cytosines (combined from both strands, adding the adjacent guanosines from the antisense-strand cytosines in CG sites) with detected methylation/demethylation events (adenosines and thymidines); their height represents the percentage of methylation in that position. The column bars (Right) show the average cytosine methylation over the entire fragment, additionally represented by a line indicating the average methylation for Ty-1 (solid) and MM (dashed). ORFs are indicated by arrows. (A) Coding region of V2 and V1. (B) Intergenic region and coding region of V2.

Fig. 4.

DNA methylation analysis of ToSRV (V1 region) isolated from MM and Ty-1 plants infected with ToSRV. Schematic overview of the multiple sequence alignment of 20 cloned PCR products per tomato plant obtained from bisulfite-treated DNA genome templates and spanning the coding region of V1. Black bars indicate the positions of cytosines (combined from both strands, adding the adjacent guanosines from the antisense-strand cytosines in CG sites) with detected methylation/demethylation events (adenosines and thymidines); their height represents the percentage of methylation in that position. ORFs are indicated by arrows. The column bars (Right) show the average cytosine methylation over the entire fragment, additionally represented by a line indicating the average methylation for Ty-1 (solid) and MM (dashed).