RNA silencing safeguards plant fertility during viral infection and decreases Turnip rosette virus vertical transmission

Aimer Gutiérrez-Díaz, Sanjana Holla, Inês Moura and Anders Hafrén*

affiliations: Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Box 7080, 75007 Uppsala, Sweden.

*correspondence: anders.hafren@slu.se

Comparative viral transcriptomic analysis

RNA-seq expression data for uninfected and infected Arabidopsis thaliana were obtained from NCBI Bioprojects: TuMV PRJNA788379 [1], TuYV and CaMV PRJEB49403 [2], TYMV PRJNA1103879 [3], TCV PRJNA336058 [4], CMV PRJNA1124548 [5] and ArLV1 PRJNA863409 [6]. Read processing, alignment, and gene-level quantification was addressed by mapping with HISAT2 v2.2.1 (Kim et al., 2019) to TAIR10 reference genome and quantifying with featureCounts (Liao et al., 2014). Differential expression was computed separately within each using DESeq2 DEGs were calculated using Deseq2 (Love et al., 2014).

Libraries re-mapped

Virus	Project	Libraries	SeqType	Tissue	Ecotype	Genotypes	dpi	Paper	Reference
ArLV1	PRJNA863409	7 (58)	Paired-End	Leaves	Col-0	WT	4% vs 80% infestation	10.1093/plphys/kiae581	[6]
TuYV	PRJEB49403	3	Paired-End	Rosette	Col-0	WT aphid Mp	14dold, 21dpi	10.1128/spectrum.00136-22	[2]
TYMV	PRJNA1103879	8 (16)	Single-End	Rosette	Col-0	WT and atg2	12dpi	10.1101/2024.05.06.590709	[3]
CaMV	PRJEB49403	6	Paired-End	Rosette	Col-0	WT aphid Mp	14dold, 21dpi	10.1128/spectrum.00136-22	[2]
TCV	PRJNA1103879	8 (16)	Single-End	Rosette	Col-0	WT and atg2	12dpi	10.1101/2024.05.06.590709	[3]
TuMV	PRJNA788379	8	Paired-End	Rosette	Col-0 and Bar-0	Wt	28dold, 14dpi	10.1371/journal.pone.0275588	[1]
CMV	PRJNA1124548	6	Paired-End	Rosette	Col-0	WT, CMV and CMV-Δ2b	14dpi	10.1038/s41467-025-65355-1	[5]

Viral RdRps structural prediction and phylogenetics

Protein sequences corresponding to CMV 2a, TRV 134K, TyMV 206K, TRoV P2ab, ALV1 P1, TuMV NIb, and PLrV, TuYV, RYMV, AhPV1, TMV, YoMV, TCV, P1AMV RdRps were curated prior to structure prediction. For viruses where the replication protein is polyprotein-derived (e.g., TRoV P2ab), sequences were processed to extract the annotated mature peptide corresponding to the RdRP-containing product, final sequences are available in this repository. Each curated protein was then folded using AlphaFold2 (Jumper et al., 2021), while CaMV P5 PBD was the only RdRp experimentally elucidated (PDB: 8R0S) [7]. To build a structure-based phylogeny, an initial structural reconstruction was performed using the predicted RdRP models, and a non-LTR retrotransposon reverse transcriptase structure (PDB: 8GH6) was included as an outgroup, in a similar way to Wolf et al. (2018). Finally, a consensus topology was obtained by performing an agreement analysis between trees generated from DALI (Holm, 2022) and Foldtree (Moi et al., 2025) outputs, retaining and scoring clades supported by both approaches.

PDB sequence post-processing

The sequence or chain selection applied to each structure in RdRps/ is summarized below. The repository contains the final PDB files but no separate processing manifest; therefore, this table reports the post-processing recoverable from filenames and coordinate records and does not infer unrecorded substitutions. Residue ranges follow the numbering stored in each PDB file. “Residues in PDB” counts residues with ATOM records; consequently, experimentally determined structures can contain fewer coordinate-bearing residues than the retained sequence span because unresolved residues are absent. Mean pLDDT was recalculated for AlphaFold2 models as the arithmetic mean of the C$\alpha$-atom B-factor field, using one value per residue. For experimental structures, this field contains experimental B-factors or related quality values rather than pLDDT and is therefore reported as not applicable (N/A).

PDB file	Protein or construct	Sequence post-processing	Retained PDB range	Residues in PDB	Mean pLDDT	Published-structure citation
ahpv1_rdrp_relax_m3_p0_plddt-90.pdb	AhPV1 RdRp	Complete submitted RdRp sequence retained; no terminal trimming	A:1–585	585	90.61	–
alv1_p1_1140_1610.pdb	ALV1 P1	N-terminal region removed to retain the C-terminal RdRp region; the stored endpoint is residue 1610	A:1140–1610	471	87.78	–
CaMV_P5_8R0S.pdb	CaMV P5 reverse transcriptase	Protein chain A retained from the experimental structure; bound nucleic-acid chains were removed	A:1–475	470	N/A	[7]
CMV_2a_273-750.pdb	CMV 2a	Internal RdRp-containing region extracted from the replication protein	A:273–750	478	88.20	–
HIV1RT_3DLK.pdb	HIV-1 reverse transcriptase	Protein chain B retained from the experimental structure	B:6–428	409	N/A	[8]
nonLTR_RT_8gh6_1_924.pdb	Bombyx mori R2 non-LTR reverse transcriptase	Protein chain A retained from the experimental structure; bound RNA and DNA chains were removed	A:111–924	715	N/A	[9]
PlAMV_RdRp_q07518_895_1385.pdb	PlAMV RdRp	C-terminal RdRp-containing region extracted from the replication protein	A:895–1385	491	85.61	–
PLrV_Polerovirus_P11623_relax_m4_p0_plddt-78.pdb	PLrV replication protein	Complete submitted replication-protein sequence retained; no terminal trimming	A:1–1062	1062	77.77	–
rymv_rdrp_1_464.pdb	RYMV RdRp	Complete submitted RdRp sequence retained; no terminal trimming	A:1–464	464	93.93	–
tcv_rdrp_relax_m1_p0_plddt-92.pdb	TCV RdRp	Complete submitted RdRp sequence retained; no terminal trimming	A:1–524	524	92.56	–
tmv_rdrp_1117_relax_m3_p0_plddt-91.pdb	TMV RdRp	C-terminal RdRp region beginning at source residue 1117 extracted and renumbered from 1 in the PDB	A:1–499	499	91.20	–
trov_p2ab_428.pdb	TRoV P2ab	N-terminal region removed to retain the mature RdRp-containing product	A:428–874	447	91.61	–
trv_134k_1206_1707.pdb	TRV 134K	C-terminal RdRp-containing region extracted	A:1206–1707	502	86.51	–
TuMV_NIb_m2_plddt-93.pdb	TuMV NIb	Mature NIb product extracted from the viral polyprotein and renumbered from 1	A:1–517	517	93.20	–
TuYV_RdRP_p09507_relax_m1_p0_plddt-80.pdb	TuYV replication protein	Complete submitted replication-protein sequence retained; no terminal trimming	A:1–1035	1035	80.63	–
tymv_206k_1298.pdb	TyMV 206K	N-terminal region removed to retain the C-terminal RdRp-containing region	A:1298–1844	547	82.12	–
YoMV_RdRP_q66220_1120_1597.pdb	YoMV RdRp	C-terminal RdRp-containing region extracted from the replication protein	A:1120–1597	478	88.25	–

TRov vs RYMV RdRp TM-align

Graphical example of the structural alignment of RdRp between the close relative Sobemovirus TRoV (Blue) and RYMV (Orange) using TM-Align algotihm from RCSB web tool:

Entry	Chain	RMSD	TM-score	Identity	Aligned Residues	Sequence Length	Modeled Residues
rymv_rdrp_1_464.pdb	A	-	-	-	-	464	464
trov_p2ab_428.pdb	A	1.32	0.94	52%	440	447	447

structural alignment of RdRp between the close relative Sobemovirus TRoV (Blue) and RYMV (Orange)

References

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