Molecular characterization of geminivirus-derived small RNAs in different plant species

Abstract

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs-the hallmarks of silencing-associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5' end phosphorylated, as shown by phosphatase treatments, and methylated at the 3'-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to beta-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions.

Figure 1

Comparison of siRNAs derived from the geminivirus ACMV and the dsRNA transgene in N.benthamiana and cassava RNA gel blot analysis of 20 µg total RNA prepared from young leaves of wild-type (lane 3) and dsRNA-transgenic (lane 4) N.benthamiana, or from ACMV-infected wild-type N.benthamiana (lane 5) or cassava (lane 6) plants. The blots were successively probed with ³²P-labeled 27 nt DNAs (Table 1) corresponding to the ACMV DNA-A complementary and virion strand sequences in the intergenic region (‘NONA as’ and ‘NONA s’, respectively) and the AC2 gene coding region (‘AC2 as’ and ‘AC2 s’). The N.benthamiana blot was stripped and re-probed with a mixture of two oligonucleotides complementary to U6 (Table 1) as a loading control. Positions of 21 and 24 nt RNAs from the in vitro synthesized, p³²-labeled size-markers (lanes 1 and 2) are indicated. Note that the extra bands appear to be the result of untemplated extension by the T7 polymerase used for marker synthesis. The contrast change between marker lanes (1 and 2) and the adjacent part of the blot is due to a difference in exposure times. Marker lanes in panels below the top one are not shown, since the marker signal decayed during successive re-probing; sizes of viral siRNAs were determined by super-imposing the corresponding images with the topmost image.

Figure 2

ACMV-derived siRNAs are phosphorylated at the 5′ end RNA gel blot analysis of 20 µg total RNA prepared from ACMV-infected wild-type N.benthamiana and treated (+) or not (−) with alkaline phosphatase. The blot was successively probed with DNA oligonucleotides corresponding to the ACMV DNA-A complementary (AC2 as) and virion (AC2 s) strand sequences in the AC2 coding region. Positions of the 21 and 24 nt RNAs are indicated.

Figure 3

ACMV- and dsRNA transgene-derived siRNAs are modified at the 3′ terminal nucleotide RNA gel blot analysis of 20 µg total RNA prepared from young leaves of wild-type (lane 3) and dsRNA-transgenic (lanes 6 and 7) N.benthamiana, or from ACMV-infected wild-type N.benthamiana (lanes 1, 2, 4 and 5) or cassava (lanes 8 and 9) and treated (+β) or not (−β) with the oxidation and β-elimination reagents. The blots were successively probed with ³²P-labeled 27 nt DNAs (see Table 1) corresponding to the intergenic region (NONA as) and the AC2 gene coding region (‘AC2 as’ and ‘AC2 s’) of ACMV DNA-A. The blots were stripped and re-probed with an oligonucleotide complementary to a synthetic RNA (‘β internal control’) added to both the samples (lanes 2, 5, 7 and 9) prior the β-elimination treatment and some non-treated control samples (lanes 1 and 3). Positions of the 21 and 24 nt RNAs are indicated.

Figure 4

DNA geminivirus-derived siRNAs are methylated in Arabidopsis, whereas a major fraction of RNA tobamovirus-derived siRNAs are not (A) RNA gel blot analysis of 20 µg total RNA prepared from the geminivirus CaLCuV-infected wild-type (La-er and Col-0) and HEN1 loss-of-function mutant (hen1-1) Arabidopsis plants, or from the tobamovirus ORMV-infected wild-type Arabidopsis, and from uninfected plants of the PTGS-silenced GFP transgenic line (GFP-PTGS), and treated (+β) or not (−β) with the oxidation and β-elimination reagents. The blots were successively probed with the virus- or the transgene-specific DNAs (‘AC2 as’, ‘ORMV’ or ‘GFP’, Table 1) and from the three endogenous loci (miR173, siR1003 and siR255). The blots were stripped and re-probed with an oligonucleotide complementary to a synthetic RNA (‘β internal control’) added to both the samples (lanes 3, 5, 7, 9 and 11) prior the β-elimination treatment and the non-treated control sample (lane 1). Positions of the 21 and 24 nt RNAs are indicated. (B) RNA gel blot analysis of 20 µg total RNA prepared from ORMV-infected N.benthamiana young leaves and treated (+β) or not (−β) with the oxidation and β-elimination reagents. The blot was probed with ORMV-specific DNAs (Table 1) and then stripped and re-probed with an oligonucleotide complementary to a synthetic RNA (‘β internal control’) added to the sample (lane 2) prior the β-elimination treatment and the non-treated control sample (lane 1). Positions of the 21 and 24 nt RNAs are indicated.

Figure 5

DCL2 and DCL3 produce distinct size-classes of CaLCuV siRNAs in Arabidopsis RNA gel blot analysis of 20 µg total RNA prepared from the geminivirus CaLCuV-infected wild-type (La-er and Col-0) and DCL2 and DCL3 loss-of-function mutant (dcl2-5 and dcl3-1) Arabidopsis plants. The blots were successively probed with ³²P-labeled DNAs specific to CaLCuV (‘AC2 as’ and ‘AC2 s’, Table 1), or complementary to endogenous ra-siRNA (siR1003) and ta-siRNA (siR255). Positions of the 21 and 24 nt RNAs are indicated.