smRNAs of ZYMV-infected squash were quantified by microarray analysis at 5 dpi. The average expression levels of selected miRNAs (miR) and miRNA*s (miR*) that were quantified by using a comprehensive smRNA microarray assay (see Fig. S1 in the supplemental material for sequences shown in Fig. 2A and B). Noninoculated squash plants (Non-Inoc.) were compared to attenuated ZYMV^FINK (FINK)- and severe ZYMV^FRNK (FRNK)-infected plants. This graphic is arbitrarily divided to present two charts of miRNA/miRNA* pairs. For the pairs presented in panel A, the concentration of FRNK miRNA* is more than threefold greater than the concentration of FINK miRNA*. For the pairs presented in panel B, the concentration of FRNK miRNA* is less than threefold greater than the concentration of FINK miRNA*. The signals were normalized in-chip to a spike-in control RNA, and chip signals were subsequently normalized to each other. The smRNAs that were labeled and hybridized were from pools of seven to nine plants for each treatment. Error bars indicate the SEMs of three independent biological repeats. The relative accumulations of smRNAs derived from the plus (C) and minus (D) strands of the ZYMV genome in ZYMV^FRNK (FRNK)- and ZYMV^FINK (FINK)-infected leaves were measured. Viral siRNAs were assayed using tiled 25-nt probes on a representative microarray, as described above, that were tiled with a 13-bp overlap. Data shown are for 193 consecutive plus-strand probes and their complementary minus-strand probes that represent the first 2,437 nt, which encode the 5′ UTR protein, P1, and HC-Pro. For clarity in panels C and D, only one of three biological replicates is shown.

The ZYMV HC-Pro FRNK motif is involved in double-stranded smRNA duplex binding. Squash leaf extracts were prepared for EMSA from ZYMV-infected (A and B) or foreign-gene-expressing, ZYMV-infected (C and D) plants. Panel A shows HC-Pro binding to an artificial miRNA/miRNA*-like duplex probe, and panel C shows HC-Pro binding to an artificial siRNA-like duplex probe. The corresponding protein-level controls for each experiment are shown in the Western blots in panels B and D. (A and B) Three leaf samples with HA-tagged HC-Pro-expressing virus and one sample with virus expressing untagged protein were collected at 5 dpi (for either severe ZYMV^FRNK- or attenuated ZYMV^FINK-infected plants), along with a noninoculated control sample (Non-Inoc.). (C and D) Representative samples from leaves inoculated with ZYMV clones that expressed GFP or CMV Fny 2b (2b) and had the indicated mutations in the FRNK motif. Bound and free radiolabeled duplexes are indicated by arrows. The radiolabeled probe alone was run as a control (duplex). § denotes a putative endogenous plant RNA duplex-binding protein that was sometimes observed in EMSA of infected and noninoculated samples. The miRNA/miRNA* duplex probe was based on the human precursor of miR16 and included mismatches and noncanonical base pairing, and the siRNA-like duplex probe had an artificial fully matching sequence with 3′ overhangs. Western blots were probed with monoclonal anti-HA antibody (B) or polyclonal anti-ZYMV HC-Pro and anti-ZYMV CP antibodies (D). Ponceau staining of the same membrane, as an equal loading control, is shown. The expected sizes of the HC-Pro monomer and the CP are marked by the arrows (50 kDa and 32 kDa, respectively).

Replacement of positively charged amino acids in two different locations of ZYMV HC-Pro reduces duplex RNA binding without abolishing RSS activity. (A) ZYMV HC^FINK and ZYMV HC^FRNA overexpressed in N. benthamiana are active as suppressors, while TEV HC^FINK is inactive. N. benthamiana leaves were agrobacterium infiltrated with different combinations of binary constructs overexpressing the different HC-Pro constructs ZYMV HC^FRNK, ZYMV HC^FINK, ZYMV HC^FRNA, TEV HC^FRNK, and TEV HC^FINK. ZYMV suppressors were HA tagged. Pictures of detached leaves were taken at 4 dpi (right panel) or 7 dpi (left panel) under UV illumination. The diagram (far right) shows the leaf infiltration pattern with binary constructs (HC-Pro construct [HC], GFP-expressing construct [GFP], GFP-silencing inverted-repeat construct [IR]). All cultures were diluted in each other or in culture with EV to an OD₆₀₀ of 0.5. (B and C) Binding assay. Leaf extracts from a separate leaf from the same plants as those in panel A were collected at 3 dpi for EMSA, which was conducted with radioactive artificial siRNA (B) or miRNA (C) duplexes. (D) The same extracts were used for Western blot analysis with the antibodies shown. The amounts of HC-Pro protein loaded per lane were estimated to be 1.7 μg for ZYMV HC^FRNK and 1.1 μg for ZYMV HC^FINK. TEV HC^FRNK protein levels were estimated to be 10-fold greater than TEV HC^FINK levels. The concentration of each probe used in the binding assays was 5 nM. § marks a putative plant-endogenous duplex-binding protein that is sometimes observed. uninoc, uninoculated.

The HC-Pro FRNK motif is highly conserved in potyviruses. Forty-four distinct potyvirus HC-Pro sequences are aligned to show a region in the central domain. The FRNK motif and two adjacent positively charged (+) and aromatic (a) residue-rich regions are underlined. The shading threshold is 80% amino acid identity. Numbering from the first amino acid of each HC-Pro is indicated. Virus proteins, extracted from viral sequences with the indicated GenBank accession numbers, are as follows: ZYMV gi 118566318 (ZYMV), Bean common mosaic necrosis virus gi 25013913 (BCMNV), Bean common mosaic virus gi 25013490 (BCMV), Bean yellow mosaic virus gi 25013500 (BYMV), Beet mosaic virus gi 40254030 (BtMV), Chilli veinal mottle virus gi 45004657 (ChiVMV), Clover yellow vein virus gi 25013510 (ClYVV), Cocksfoot streak virus gi 25014039 (CSV), Cowpea aphid-borne mosaic virus gi 25013520 (CABMV), Daphne virus Y gi 96980663 (DVY), Dasheen mosaic virus gi 25013784 (DsMV), East Asian passiflora virus gi 85539887 (EAPV), Japanese yam mosaic virus gi 25013883 (JYMV), Johnsongrass mosaic virus gi 25013809 (JGMV), Konjak mosaic virus gi 90093254 (KoMV), Leek yellow stripe virus gi 25013893 (LYSV), Lettuce mosaic virus gi 25013530 (LMV), Lily mottle virus gi 39163617 (LMoV), Maize dwarf mosaic virus gi 25013540 (MDMV), Onion yellow dwarf virus gi 32493289 (OYDV), Papaya leaf distortion mosaic potyvirus gi 32493279 (PLDMV), Papaya ringspot virus gi 25013550 (PRSV), Pea seed-borne mosaic virus gi 25013560 (PSbMV), Peanut mottle virus gi 25013833 (PeMoV), Pepper mottle virus gi 25013570 (PepMoV), Peru tomato mosaic virus gi 28519942 (PTV), PPV gi 25013580 (PPV), Potato virus A gi 25013590 (PVA), Potato virus V gi 25013850 (PVV), PVY gi 25013600 (PVY), Scallion mosaic virus gi 25013997 (SVY), Shallot yellow stripe virus gi 76803358 (SYSV), Sorghum mosaic virus gi 25013823 (SrMV), Soybean mosaic virus gi 25013610 (SMV), Sugarcane mosaic virus gi 25013620 (SCMV), Sweet potato feathery mottle virus gi 25013774 (SPFMV), Thunberg fritillary virus gi 68989219 (TFV), TEV gi 25013634 (TEV), Tobacco vein mottling virus gi 25013639 (TVMV), TuMV gi 25013650 (TuMV), Watermelon mosaic virus gi 51949948 (WMV), Wild potato mosaic virus gi 25141239 (WPMV), Wisteria vein mosaic virus gi 116723233 (WVMV), and Yam mosaic virus gi 48249198 (YMV).

A mutation in the FRNK motif of HC-Pro attenuates symptoms without reducing initial HC-Pro levels. (A) Squash (cv. Ma'ayan) leaves showing systemic symptoms at 5, 7, and 14 dpi with either severe (ZYMV^FRNK) or attenuated (ZYMV^FINK) ZYMV infection. Noninoculated leaves (Non-Inoc.), as a control, are shown. (B) The levels of HA-tagged HC-Pro and CP at 5 dpi were measured by Western blotting by using the antibodies indicated. A noninoculated leaf sample (NI) was loaded as a control. Arrows indicate the sizes of the nearest protein marker (M) bands. Ponceau staining was used to demonstrate equal protein loading.

Accumulations of miRNAs and miRNA*s 159, 166, and 168 in ZYMV-infected squash leaves are differently modulated over time by a mutation in the FRNK motif. RNA filter hybridization of noninoculated (H), attenuated ZYMV^FINK-infected (FI), and severe ZYMV^FRNK-infected (FR) squash RNA with antisense probes to the smRNAs indicated (see Fig. S1 in the supplemental material for the sequences). Ten or 20 μg of total RNA that was isolated from eight plants per treatment was loaded per lane. Days postinfection (dpi) are indicated at the top of the figure. Ethidium bromide (EtBr) staining in the tRNA zone is shown as an equal-loading control for one of the six similar gels.

The suppressor activities of attenuated forms of HC-Pro are not compromised. N. benthamiana was agrobacterium infiltrated with equal culture concentrations with different combinations of binary constructs expressing GFP, a GFP inverted repeat (IR), the HA-tagged ZYMV HC-Pro constructs HC^FRNK (wild type) and HC^FINK (attenuated), or the EV. HC-Pro-expressing cultures were also diluted 20-fold (1/20) in EV-containing culture, while the GFP and IR concentrations were held constant. (A) Photographs were taken under UV light at 4 days postinfiltration, and protein and RNA samples were taken at the same time. (B) HC-Pro and GFP expression levels were detected by Western blotting in equal amounts of protein extract, probed with anti-HA or with anti-GFP monoclonal antibodies. The expected sizes of the HC-Pro monomer and GFP are marked by the arrows (50 kDa and 27 kDa, respectively). (C) GFP siRNA accumulation was detected by use of smRNA Northern blots containing 10 μg of total RNA per lane. The membrane was hybridized with radiolabeled sense-strand GFP RNA transcript. Size was assessed with DNA oligonucleotides stained with ethidium bromide (EtBr). The EtBr-stained gel for the tRNA zone is shown as an equal-loading control.

ZYMV HC^FRNK binds an siRNA-like duplex with a much greater affinity than ZYMV HC^FINK. (A) A dilution series of N. benthamiana leaf extract expressing ZYMV HC^FRNK was assayed in an EMSA binding reaction with 4.76 nM siRNA-like duplex. A representative EMSA gel is shown (inset). Samples were diluted in EV-infiltrated leaf extracts to the HC-Pro concentrations indicated. The Hill plot (line graph) was derived from the averages of four such experiments. Signals were quantified from phosphorimager data using ImageJ software. The Hill plot and biochemical values (top left) were generated using Origin 7.5 software. Bars show standard deviations for the four replicates. n, apparent Hill coefficient. (B) Electromobility shift competition assay. γ-³²P-radiolabeled siRNA-like duplex was premixed to 4.54 nM with different concentrations of nonradioactive phosphorylated competitor duplex (cold) and incubated with N. benthamiana extracts expressing HA-tagged ZYMV HC^FRNK or HC^FINK. Competition assays were conducted with undiluted (2 μM) HC^FINK-expressing extract (B) and at a 50-fold dilution (in uninoculated leaf extract) for HC^FRNK (60 nM) (C). Extract from noninoculated leaves (neg) and RNA duplex without extract (dup) were run as controls. § marks a putative plant endogenous duplex-binding protein sometimes seen in EMSA.

Model of HC-Pro influence on symptom expression. miRNAs are processed from a hairpin precursor (pre-miRNA) by a DICER-like enzyme (DCL1) to make duplex miRNA/miRNA*. In uninfected plants, miRNA is incorporated into the RISC, and miRNA* is degraded. RISC then down-regulates the expression of a target mRNA, complementary to the miRNA, through cleavage. In potyvirus-infected plants, HC-Pro binds the miRNA/miRNA* duplex, effectively sequestering miRNA and causing ectopic expression of target mRNAs. In wild-type HC-Pro, the FRNK motif contains an aromatic amino acid residue (a) and two positively charged amino acid residues (+) which are involved in duplex binding. Charge reduction in the ZYMV^FINK-attenuated virus causes lower affinity for miRNA/miRNA* duplexes, especially for those which contain bulges or multiple mismatches, allowing adequate RISC function and, thus, mild symptom expression.