IGP by <i>Harmonia axyridis</i> on <i>Episyrphus balteatus</i> on potted pepper plants after 24h.

<p>Percentage of third instars of <i>Episyrphus balteatus</i> reared on an <i>ad libitum</i> or limited supply of aphids that were killed through IGP on pepper plants after 24h by third instars of <i>Harmonia axyridis</i> reared on various diets. Aph ad lib: <i>ad libitum</i> supply of <i>Acyrthosiphon pisum</i> aphids, Aph lim: limited supply of aphids, Pollen: moist honey bee pollen. No statistically significant effect of larval diet of <i>H</i>. <i>axyridis</i> was found (generalized linear model with Poisson error distribution and a log link function). Error bars represent SE-values.</p

Total developmental time of <i>Episyrphus balteatus</i> immatures (from first instar to adulthood), weight of the third instar, survival time under starvation of third instars and 6h predation rate by third instars (% of <i>Acyrthosiphon pisum</i> aphids killed) when offered various diets.

<p>Values in the same column followed by a different letter are significantly different. n: sample size.</p><p>Total developmental time of <i>Episyrphus balteatus</i> immatures (from first instar to adulthood), weight of the third instar, survival time under starvation of third instars and 6h predation rate by third instars (% of <i>Acyrthosiphon pisum</i> aphids killed) when offered various diets.</p

Total developmental time of <i>Harmonia axyridis</i> immatures (from first instar to adulthood), weight of the fourth instar, survival time under starvation of third instars and 6h predation rate by third instars (% of <i>Acyrthosiphon pisum</i> aphids killed) when offered various diets.

<p>Values in the same column followed by a different letter are significantly different. n: sample size.</p><p>Total developmental time of <i>Harmonia axyridis</i> immatures (from first instar to adulthood), weight of the fourth instar, survival time under starvation of third instars and 6h predation rate by third instars (% of <i>Acyrthosiphon pisum</i> aphids killed) when offered various diets.</p

IGP by <i>Harmonia axyridis</i> on <i>Episyrphus balteatus</i> in Petri dishes after 90 minutes.

<p>Percentage of third instars of <i>Episyrphus balteatus</i> reared on an <i>ad libitum</i> or limited supply of aphids that were killed through IGP in Petri dishes after 90 minutes by third (A) and fourth (B) instars of <i>Harmonia axyridis</i> reared on various diets. Eph: <i>Ephestia kuehniella</i> eggs, Aph ad lib: <i>ad libitum</i> supply of <i>Acyrthosiphon pisum</i> aphids, Aph lim: limited supply of aphids, Pollen: moist honey bee pollen. The effect of larval diet of <i>H</i>. <i>axyridis</i> is indicated by lowercase letters (generalized linear model with Poisson error distribution and a log link function); within each panel, groups of bars followed by a different letter are significantly different. Error bars represent SE-values.</p

Number of contacts (sum of the contacts that led to an attack and those without consequences) during the first 90 min of the Petri dish IGP experiments, number of contacts that resulted in an attack by <i>Harmonia axyridis</i> or <i>Episyrphus balteatus</i> and total number of replicates in which the attack by <i>H</i>. <i>axyridis</i> was successful (n = 20) for each combination of larvae reared on the various diets (means ± SE). L3–L4: third and fourth instar of <i>H</i>. <i>axyridis</i>.

<p>Number of contacts (sum of the contacts that led to an attack and those without consequences) during the first 90 min of the Petri dish IGP experiments, number of contacts that resulted in an attack by <i>Harmonia axyridis</i> or <i>Episyrphus balteatus</i> and total number of replicates in which the attack by <i>H</i>. <i>axyridis</i> was successful (n = 20) for each combination of larvae reared on the various diets (means ± SE). L3–L4: third and fourth instar of <i>H</i>. <i>axyridis</i>.</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

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

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

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

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