CY2 and CY2sgm1 accumulated in phloem tissues of stem, petiole, and roots.

FISH analysis of CY1, CY2, and CY2sgm1 at 60 dpi. Cy3-labeled oligonucleotide probe targeted CY1 and CY2 positions 1101–1131. DAPI-stained DNA and xylem tissue fluoresce blue. Bar = 50 μm. No viral RNA signals were detected in non-phloem tissue. (PDF)</p

CY1 systemically infects cucumber.

(A) Schematic representation of a cucumber plant (Cucumis sativus cv. Spacemaster) agroinfiltrated with CY1 on cotyledons (solid green). Systemic leaves (numbered 1 to 4) were used to test for the presence of CY1. (B) Representative RT-PCR detection of CY1 in systemic leaves of 4 cucumber plants (Cs-1 to Cs-4) at 14 dpi. Negative control was the reaction in the absence of template and positive control was plasmid DNA containing full-length CY1. (C) Strand-specific detection of (+)- and (-)-strand CY1 in systemic leaves of 8 cucumber plants infiltrated with CY1 at 21 dpi. (-)-strand and (+)-strand lanes used strand-specific primers and plasmid DNA containing full-length CY1. (PDF)</p

Percentage of <i>N</i>. <i>benthamiana</i> plants showing symptoms at different times post-infiltration of CY1, CY2, and CY2 ORF5 mutants.

Percentage of N. benthamiana plants showing symptoms at different times post-infiltration of CY1, CY2, and CY2 ORF5 mutants.</p

Raw images of all gels can be found at https://figshare.com/search?q=10.6084/m9.figshare.25407199.

Raw images of all gels can be found at https://figshare.com/search?q=10.6084/m9.figshare.25407199.</p

ORF5_CY2 forms VLPs in infected <i>N</i>. <i>benthamiana</i>.

(A) CY1- and CY2-infected plants were subjected to ultracentrifugation to gently purify any VLPs. Material from CY1- and CY2-infected N. benthamiana in the 70% sucrose cushion, interface (Int), and 25% sucrose cushion was examined following electrophoresis through a 0.8% agarose gel. Nb, N. benthamiana. (B) Western blot detection of ORF5CY2. Total proteins from healthy N. benthamiana and CY2-infected N. benthamiana, and 70% sucrose fractions of CY1- and CY2-infected N. benthamiana were separated by 10% PAGE. ORF5CY2-specific antibody was used to detect ORF5CY2 (arrow). (C) Northern blot detection of CY2 gRNA and sgRNA in material from the 70% sucrose fraction of CY2-infected N. benthamiana. Total RNA from uninfected N. benthamiana and CY2-infected N. benthamiana served as negative and positive controls, respectively. (D) Samples of 70% sucrose cushions from uninfected N. benthamiana, CY1-infected N. benthamiana, and CY2-infected N. benthamiana were exchanged to PBS buffer and observed by EM. Particles of 3 different sizes were found in CY2-infected N. benthamiana that were absent from uninfected and CY1-infected N. benthamiana. Yellow arrows point to CY2-infected N. benthamiana particles with a diameter of 14 nm that were the most prevalent. Red arrow denotes a 24 nm particle and blue arrow denotes a 35 nm particle. EM, electron microscopy; N b., N. benthamiana; ORF, open reading frame; VLP, virus-like particle.</p

CY2 replicates and moves systemically in the absence of ORF5.

(A) Secondary structures of a portion of CY1 (left) and the corresponding region of CY2 (right). Sequence shaded light green in CY2 is a “carmovirus consensus sequence” (G2-3A/U5-9) located at the 5′ ends of carmovirus and umbravirus gRNAs and sgRNAs. Red residues differ between CY1 and CY2. ORF5 initiation codon is circled and shaded yellow. CY2 mutants CY2sgm1 and CY2sgm2 have color-coded alterations in the sgRNA promoter region. CY25T contains a truncated ORF5 protein (33 amino acids remaining). (B) Accumulation of (+)- and (-)-strands of CY1, CY2, and CY2 mutants in Arabidopsis protoplasts at 24 h and 48 h post-transfection assayed by northern blots. CY1 accumulation was low but detectable. CY1 with an RdRp active site alteration (CY1-GDD) served as a negative control. Mock, no added RNA. Data was obtained from 3 independent experiments. Standard deviation is shown. No significant difference in accumulation of the gRNA was found for CY2 and the CY2 mutants using JMP Pro 16, Student’s t, α = 0.05. (C) Competition assays between CY1 and WT and mutant CY2. Symptomatic systemic leaves were collected at 3-, 4-, and 6-wpi and total RNA was extracted and subjected to northern analysis. CY1 and CY2 size differences were used to visually assess levels of the different gRNAs. Three independent experiments were conducted with very similar results. Note that while WT CY2 is mainly detected at 3-wpi in co-infiltrated plants, both CY1 and CY2 are present at 4- and 6-wpi (left panel). In contrast, CY1 is mainly detected at all time points in plants co-infiltrated with the ORF5 deficient or defective mutants (middle and right panels). ●, denotes low level band that is not detected in CY2sgm1 protoplasts infections that is near the size of the sgRNA (831 nt) and is also the size of a defective (D)-RNA (921 nt) that commonly arises in CY1/CY2 infections. ORF, open reading frame; RdRp, RNA-dependent RNA polymerase; wpi, weeks post-infiltration; WT, wild type.</p

CY1-mediated silencing of <i>PP2</i> mRNA results in less severe symptoms on <i>N</i>. <i>benthamiana</i>.

Photos of 4 plants each infected with WT CY1, CY1-random, and CY1-PP2 were taken at 50 dpi. (PDF)</p

CY1 systemically infected Mexican lime in the absence of a helper virus.

(A) Two approaches that were used to infect Mexican lime with CY1. Upper panel, two-week-old N. benthamiana were infiltrated with Agrobacterium containing a CY1-expressing Ti plasmid [step 1]. Fourteen days after infiltration, CY1-positive plants were colonized by dodder vines [step 2]. After detection of CY1 in dodder vines by RT-PCR, dodder tips were guided to colonize 2-month-old Mexican lime [step 3]. Lower panel, leaves and stems of 2-month-old Mexican lime were abraded using a Derma microneedle roller (Amazon:B0CH7SHD5T), and then vacuum-infiltrated with Agrobacterium containing a CY1-expressing Ti plasmid. (B) Right, northern blot used to detect CY1 in Mexican lime 15 months after dodder transfer or 12 months after vacuum infiltration. Left, infected plants at these times, and subsequently, did not display discernable systems. N.b., Nicotiana benthamiana; M.L., Mexican lime. (PDF)</p

ORF5_CY2 is structurally similar to 30K MPs and luteovirus/polerovirus/sobemovirus CPs.

(A) Structure modeling of full-length ORF5CY2 using Alphafold2. Color coding from blue to red indicates high to low structural confidence, respectively. Unstructured N-terminal region contains a predicted nucleolar localization signal (NoLS). (B) Overlay images of ORF5CY2-GFP and RFP-NoLS in N. benthamiana. Leaves were co-infiltrated with Agrobacteria expressing ORF5CY2-GFP and RFP-NoLS from CaMV 35S promoters. Infiltrated leaves were collected at 2 dpi and subjected to confocal microscopy. (C) Single jelly-roll domains of ORF5CY2, OuMV MP (MPOuMV), and BYDV P3 CP (CPBYDV). Full-length structures of MPOuMV and CPBYDV are boxed. CPBYDV also contains an N-terminal NoLS. (D) Maximum-likelihood phylogenetic tree based on amino acid sequences of Class 2 ORF5 (blue), representative 30K MPs (red), and SeMV (sobemovirus), BYDV (luteovirus), and PLRV (polerovirus) CPs (green). Branch numbers indicate bootstrap support in percentage out of 1,000 replicates. The letters beneath the branch numbers indicate the node names. The scale bar denotes amino acid substitutions per site. The tree is mid-point rooted. Protein sequences were aligned through PROMALS3D and subjected to phylogenetic analysis in MEGA X. BYDV, barley yellow dwarf virus; CP, capsid protein; dpi, days post-infiltration; MP, movement protein; ORF, open reading frame; OuMV, ourmia melon virus; PLRV, potato leafroll virus; SeMV, sesbania mosaic virus.</p

CY1 systemically infects <i>N</i>. <i>benthamiana</i> in the absence of a helper virus.

(A) Leaves 3 and 4 were agroinfiltrated at the 4-leaf stage with a Ti plasmid expressing full-length CY1 transcripts. Initial heart-shaped leaf (leaf 9; black arrow) was visible at 21 dpi. Subsequent emerging leaves were small and curled down at the margins (red arrow). (B) Typical phenotype of infected plants at 50 dpi. Multiple leaves emerged at each node and roots contained multiple galls. Infected plants were stunted compared with uninfected plants of the same age (right panel). Infected plants senesced within 3 to 4 months post-infiltration. (C) Detection of CY1 in systemic upper leaves by northern blot at 19 dpi. Total RNA was extracted from specified leaves (leaf numbering shown in A) and subjected to northern blot analysis. Ethidium-bromide stained rRNA was used as a loading control. (D) Stem and 3 sections of the root gall were excised from a 50 dpi plant (shown in left panel) and pressed onto nitrocellulose, which was probed for CY1 (right panel). G1-3, galls. dpi, days post-infiltration.</p