The Ability of PVX p25 to Form RL Structures in Plant Cells Is Necessary for Its Function in Movement, but Not for Its Suppression of RNA Silencing

<div><p>The p25 triple gene block protein of <em>Potato virus X</em> (PVX) is multifunctional, participating in viral movement and acting as a suppressor of RNA silencing. The cell-to-cell movement of PVX is known to depend on the suppression function of p25. GFP-fused p25 accumulates in rod-like (RL) structures with intense fluorescence in cells. By monitoring the location of fluorescence at different times, we have now shown that the RL structure is composed of filaments. P25 mutants without the conditional ability to recover movement function could not form RL structures while the mutants that had the ability did form the structure, suggesting that the ability of p25 to form RL structures is necessary for its function in cell-to-cell movement, but not for its suppressor function. Moreover, chemical inhibition of microfilaments in cells destroyed the formation of the complete RL structure. Additionally, TGBp2 and TGBp3 were recruited into the RL structure, suggesting a relationship between the TGBps in virus movement.</p> </div

Effect of chemical inhibition of microfilaments or microtubules on the formation of the RL structure.

<p>At 5–20 µM, LatB, an inhibitor of microfilaments, inhibits the formation of the complete RL structure, but not the formation of primary RL filaments. After formation of the RL structure, LatB treatment has no effect. Treatment with Oryzalin, an inhibitor of microtubules, has no effect on the formation of the structure either before or after the structure is formed. Scale bar, 50 µm.</p

Investigating the RL structure of the reported p25 mutants.

<p>Upper panels: twelve single amino acid mutants of p25 were fused with GFP and expressed in epidermal cells. An RL structure is visible in cells expressing T117A, P122S or Y221H fused with GFP, but not in cells expressing the other GFP-fused mutants. Lower panels: Recovery analysis of viral movement with mutant P122S. Fluorescence diffuses into the neighboring cells in nearly all the loci when P122S and PVX-GFPΔp25 (pGR106) are co-expressed together with p19 (bottom left), suggesting that P122S can recover the movement of PVX-GFPΔp25 when co-expressed with the suppressor p19. The recovery is weak compared to the wild-type control (p25/pGR106, bottom right) but nevertheless helps virus move into limited layers of neighboring cells (average about 200 µm) compared to the negative control (pGus/pGR106/p19). Scale bar, 50 µm.</p

TGBp2 and TGBp3 are aligned on the RL structure.

<p>A shows the co-location of RFP fused-TGBp2 and GFP-fused p25 at different scales. B shows the co-location of RFP fused-TGBp3 and GFP-fused p25 at different scales. Scale bar, 20 µm.</p

Diagram showing the plasmids used in this work.

<p>Plamsids were constructed into the pCV1300 binary vector that was developed from pCAMBIA1300 with the CaMV 35S promoter.</p

Identification of Novel <em>Oryza sativa</em> miRNAs in Deep Sequencing-Based Small RNA Libraries of Rice Infected with <em>Rice Stripe Virus</em>

<div><p>MicroRNAs (miRNAs) play essential regulatory roles in the development of eukaryotes. Methods based on deep-sequencing have provided a powerful high-throughput strategy for identifying novel miRNAs and have previously been used to identify over 100 novel miRNAs from rice. Most of these reports are related to studies of rice development, tissue differentiation, or abiotic stress, but novel rice miRNAs related to viral infection have rarely been identified. In previous work, we constructed and pyrosequenced the small RNA (sRNA) libraries of rice infected with <em>Rice stripe virus</em> and described the character of the small interfering RNAs (siRNA) derived from the RSV RNA genome. We now report the identification of novel miRNAs from the abundant sRNAs (with a minimum of 100 sequencing reads) in the sRNA library of RSV-infected rice. 7 putative novel miRNAs (pn-miRNAs) whose precursor sequences have not previously been described were identified and could be detected by Northern blot or RT-PCR, and were recognized as novel miRNAs (n-miRNAs). Further analysis showed that 5 of the 7 n-miRNAs were up-expressed while the other 2 n-miRNAs were down-expressed in RSV-infected rice. In addition, 23 pn-miRNAs that were newly produced from 19 known miRNA precursors were also identified. This is first report of novel rice miRNAs produced from new precursors related to RSV infection.</p> </div

Putative novel miRNAs produced from potential new precursor sequences not previously described.

<p>a: Sequencing reads (SR) were normalized to one million with the unique sequence reads of each library.</p><p>b: exp1 is the experiment reported by us. Sequencing reads in the non-infected sRNA library are bracketed, while those in the RSV-infected sRNA library are not.</p><p>c: exp2-4 are the three repeats of Du et al (2011). Sequencing reads in non-infected sRNA libraries are bracketed, while those in RSV-infected sRNA libraries are not.</p><p>d: mean of the number of infected divided by number of non-infected reads; the following columns show the standard deviation and the <b><i>t-</i></b>test value to test whether the mean differs significantly from 1 (equal numbers of reads).</p>*<p>: indicating the sRNAs that have the significant changes in RSV-infected rice (<i>p</i> value<0.05).</p

The putative novel miRNAs newly produced from known miRNA precursors.

<p>a: Sequencing reads (SR) were normalized to one million with the unique sequence reads of each library.</p><p>b: exp1 is the experiment reported by us. Sequencing reads in the non-infected sRNA library are bracketed, while those in the RSV-infected sRNA library are not.</p><p>c: exp2–4 are the three repeats of Du et al (2011). Sequencing reads in non-infected sRNA libraries are bracketed, while those in RSV-infected sRNA libraries are not.</p><p>d: mean of the number of infected divided by number of non-infected reads; the following columns show the standard deviation and the <b><i>t-</i></b>test value to test whether the mean differs significantly from 1 (equal numbers of reads).</p>*<p>: indicating the sRNAs that have the significant changes in RSV-infected rice (<i>p</i> value<0.05).</p

Known miRNAs identified from the sRNA library of RSV-infected rice.

<p>Known miRNAs identified from the sRNA library of RSV-infected rice.</p

sRNAs that were recognized as newly produced miRNAs from known precursors and their location in the secondary structures of their precursors.

<p>The sequences of the known mature miRNAs in precursors are colored with blue. Names, sequences and sequencing reads (bracketed) of sRNAs identified here are colored with green. The location of sRNA is shown by a black line. Mismatched nucleotides are shown in red.</p