Combined activity of DCL2 and DCL3 is crucial in the defense against potato spindle tuber viroid

Viroids are self replicating non-coding RNAs capable of infecting a wide range of plant hosts. They do not encode any proteins, thus the mechanism by which they escape plant defenses remains unclear. RNAi silencing is a major defense mechanism against virus infections, with the four DCL proteins being principal components of the pathway. We have used Nicotiana benthamiana as a model to study Potato spindle tuber viroid infection. This viroid is a member of the Pospiviroidae family and replicates in the nucleus via an asymmetric rolling circle mechanism. We have created knock-down plants for all four DCL genes and their combinations. Previously, we showed that DCL4 has a positive effect on PSTVd infectivity since viroid levels drop when DCL4 is suppressed. Here, we show that PSTVd levels remain decreased throughout infection in DCL4 knockdown plants, and that simultaneous knockdown of DCL1, DCL2 or DCL3 together with DCL4 cannot reverse this effect. Through infection of plants suppressed for multiple DCLs we further show that a combined suppression of DCL2 and DCL3 has a major effect in succumbing plant antiviral defense. Based on our results, we further suggest that Pospoviroids may have evolved to be primarily processed by DCL4 as it seems to be a DCL protein with less detrimental effects on viroid infectivity. These findings pave the way to delineate the complexity of the relationship between viroids and plant RNA silencing response

PSTVd infectivity in F1 DCLi crosses.

<p>PSTVd infected plants were analyzed at 3wpi. (A) Representative northern blots of (a) DCL2.11(x)3.10i, (b) DCL2.11(x)3.1i, (c) DCL3.10(x)2.41i and (d) DCL3.10(x)2/4.5i, DCL2/4.5(x)3.10i crosses. Total RNA staining (methylene blue) was used as loading control. Northern blots were quantified with Quantity One 4.4.1 software and are presented in (B). ‘n’, number of plants quantified. Student <i>t</i>-test was performed and significance levels were set as following: p<0.05 (*), p<0.01 (**) and p<0.001 (***).</p

vd-siRNAs profiling in DCLi single or crossed plant lines.

<p>Small RNAs from 3 weeks infected <i>N</i>. <i>benthamiana</i> plants were analyzed in polyacrylamide gels and 21, 22 and 24nt of PSTVd were monitored. U6 was used as a loading control. In panel (A) DCLi single knock-down lines and in (B) DCLi F1 crosses. Quantification of small RNAs were made using Quantity One 4.4.1 shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005936#ppat.1005936.s008" target="_blank">S2 Table</a>.</p

Proposed model of DCL involvement in PSTVd defense.

<p>In WT, PSTVd is targeted by at least three DCL enzymes. It is not clear whether DCL1 also contributes to this targeting. 21 and 22nt vd-siRNAs are loaded into AGO1 and AGO2 whereas 24nt into AGO4 and AGO5. These vd-siRNAs are probably redirected to target the PSTVd genome. In DCL4i condition, DCL4 is strongly suppressed, thus PSTVd genome is processed preferentially by a combination of DCL2-DCL3. The contribution of DCL1 in this action remains open. The majority of vd-siRNA produced at this condition are of 22nt class followed by 24nt class. These are probably uptaken by AGO1, AGO2, AGO4 and AGO5 and are targeting the PSTVd genome in a highly efficiently manner, resulting in a strong reduction of PSTVd infectivity.</p

<i>N</i>. <i>benthamiana</i> DCLi plants have reduced levels of all targeted DCLs.

<p>(A) Schematic representation of <i>N</i>. <i>benthamiana</i> DCL proteins (based on information from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005936#ppat.1005936.ref010" target="_blank">10</a>]). Abbreviations stand for: <u>DEAD-DEAH</u>-DEAD box helicases, <u>Helicase</u>-Helicase C, <u>DSRD</u>-double-stranded RNA-binding domain, <u>PAZ</u>-Piwi Argonaute Zwille, <u>RNAse III</u>-Ribonuclease II, <u>DSRM</u>-double-stranded RNA-binding motif. The orange box represents the domain used to design the hairpins. The blue lines correspond to the selected fragment for qPCR analysis. (B) qPCR analysis of all DCLi single lines (n = 6). (C) qPCR analysis of F1 crosses line plants (n = 3–5) (a) DCL1.13(x)2.11i, (b) DCL1.13(x)3.10i, (c) DCL1.13(x)4.9i, (d) DCL2.11(x)3.10i, (e) DCL2/4.5i and DCL2/4.16i, (f) DCL4.9(x)3.10i, (g) DCL3.10(x)2/4.5i and (h) DCL2/4.16(x)1.13i.</p

Further analysis of <i>Pospoviroidae</i> infections in DCL4i plants.

<p>(A) Time course of PSTVd infection in WT and DCL4.9i plants. Three plants were tested and a representative northern blot is presented. (B) Infections of WT and DCL4.9i plants with PSTVd^KF493732.1, TASVd^KF484878.1 (both at 5wpi) and HSVd^Y09352 (3wpi). (C) Detection of (-) and (+) strand RNAs of PSTVd in WT and DCL4.9i plants and of HSVd in the same plants (D). In all northern blots, total RNA staining (methylene blue) was used as loading control. Quantification of northern blots was performed with Quantity One 4.4.1. Graphical representation and statistical analysis with Graphpad Prism 6 are presented in (b). ‘n’ corresponds to the number of individual plants tested. Results were analyzed with unpaired Student <i>t</i>-test, and the level of significance was set as p<0.01(**) and p<0.001 (***).</p

PSTVd infectivity in DCL1.13(x)4.9i and DCL4.9(x)3.10i plants.

<p>(A) Representative northern blots of DCL1.13(x)4.9i and DC4.9(x)3.10i PSTVd infected plants at 3wpi. Hybridizations were performed with DIG labeled (-) RNA strand of PSTVd, and total RNA staining (methylene blue) was used as internal loading control. (B) Graphical presentation of the infectivity quantification of PSTVd. Quantification was made using Quantity One 4.4.1 software. Student <i>t</i>-test was performed and significance level was set to p<0.05 (*). ‘n’ stands for the number of individual plants tested.</p

DCL levels upon PSTVd infection.

<p>(A) Volcano plot from microarray experiment with RNA from leaves of WT and PSTVd <i>N</i>. <i>benthamiana</i> infected plants (3wpi). <i>N</i>. <i>benthamiana</i> genes with no significant alteration of their expression level are indicated with black dots, while red dots represent genes with a significant higher or lower expression (fold change (FC) ≥ 2, Benjamini-Hochberg (BH) FDR-corrected P-value < 0.05) in PSTVd infected plants compared to WT plants. The 35 <i>N</i>. <i>benthamiana</i> RNAi components (described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005936#ppat.1005936.ref010" target="_blank">10</a>]) are indicated with green dots. The PSTVd virus sequence and its reverse complement (RC) are both labeled (see micorarray design) (B) qPCR experiments of DCL transcripts upon infection. Two reference genes (L23, FBOX) were used for normalization. No significant differences were observed.</p

PSTVd infectivity in F1 DCLi crosses.

<p>PSTVd infected plants were analyzed at 3wpi. (A) Representative northern blots of (a) DCL2.11(x)3.10i, (b) DCL2.11(x)3.1i, (c) DCL3.10(x)2.41i and (d) DCL3.10(x)2/4.5i, DCL2/4.5(x)3.10i crosses. Total RNA staining (methylene blue) was used as loading control. Northern blots were quantified with Quantity One 4.4.1 software and are presented in (B). ‘n’, number of plants quantified. Student <i>t</i>-test was performed and significance levels were set as following: p<0.05 (*), p<0.01 (**) and p<0.001 (***).</p