Groen et al JIP 2015 Raw Data

Raw data from the laboratory experiments described in Groen et al. (Journal of Insect Physiology, 2015). Details on experimental conditions can be found in this manuscript

A Trio of Viral Proteins Tunes Aphid-Plant Interactions in<i>Arabidopsis thaliana</i>

<div><p>Background</p><p>Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of <i>Arabidopsis thaliana</i>, one of the natural hosts for CMV, with <i>Myzus persicae</i> (common names: ‘peach-potato aphid’, ‘green peach aphid’). </p> <p>Methodology/Principal Findings</p><p>Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0. </p> <p>Conclusions/Significance</p><p>Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences.</p> </div

Induction of <i>GUS</i> reporter genes fused to the promoters of MYB51<i>, CYP79B2</i>, and <i>CYP81F2</i> in response to Fny-CMV infection at 14 and 21 days post-inoculation (dpi).

<p>Three plants per treatment group, per time-point were analyzed and photographs were taken of representative plants. Scale bars represent 1 cm.</p

Virus-induced anti-aphid resistance mapped to Fny-CMV RNA2.

<p>(A) Growth rate of individual aphids on plants infected with reassortant versions of CMV, n≥24. (B) Accumulation of viral RNA measured by RT-Q-PCR relative to the accumulation of Fny-CMV (F₁F₂F₃) at 14 days post-inoculation. Viral reassortants generated using combinations of CMV genomic RNAs 1, 2 or 3 (subscript numbering) of either Fny-CMV (indicated by F) or LS-CMV (L) were used to inoculate wild-type Arabidopsis plants (Col-0 ecotype). Mock indicates a mock-inoculated plant. Different letters (a-d) are assigned to statistically different results (ANOVA with post-hoc Tukey’s tests, <i>P</i><0.05). Error bars represent standard error of the mean.</p

Disruption of AGO1 function by Fny2b induced antibiosis but not feeding deterrence to aphids.

<p>(A, D) Growth rate of individual aphids feeding on transgenic plants expressing the Fny2b or LS2b proteins and <i>ago1-25</i> mutant plants, which are compromised in AGO1 activity, a major molecular target of Fny2b, n≥24. (B, E) Electrical penetration graph analysis of the amount of phloem feeding in <i>Fny2b</i>-transgenic plants and <i>ago1-25</i> mutants over a 12-hour recording period, broken down into hours 1-6, 7-12, and over the entire period (C, F) Results of host-switching experiments where aphids were moved from their initial hosts (“before switch”) to uninfected, wild-type plants (“after switch”) to see if aphids could recover from any effects of the initial host on aphid growth rate, n≥24. Results of Student’s <i>t</i>-tests are indicated by asterisks: *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. Error bars represent standard error of the mean.</p

Aphid behavior and performance on virus-infected wild-type Arabidopsis plants.

<p>(A) Ten aphids (<i>Myzus persicae</i>) were released onto rosettes of mock-inoculated or Fny-CMV-infected release plants and then allowed to remain or emigrate to a plant of the opposite treatment group located 10 cm away in the same pot. Aphids migrated away more often from Fny-CMV-infected than from mock-inoculated plants. Fewer aphids remained on Fny-CMV-infected than on mock-inoculated release plants after 24 hours. Based on the methods of Mauck et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083066#B8" target="_blank">8</a>], three independent tests were performed for each type of release plant. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083066#pone.0083066.s001" target="_blank">Figure S1</a> for the accompanying aphid choice data. (B) Mean relative growth rate of individual aphids feeding on Arabidopsis plants infected with Fny-CMV, n≥24. Error bars represent standard error of the mean. Asterisks indicate significant differences (Student’s <i>t</i>-test): *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Hypothesis: Differential effects of virus infection on aphid plant interactions may favor either transmission or persistence.

<p>CMV infection can engender deterrence to prolonged aphid feeding on certain hosts (Type 1), which discourages settling and provides an incentive to move from host to host (indicated by arrowed circle) (Reviewed in reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083066#B4" target="_blank">4</a>]). Aphid feeding behavior on Type 1 hosts is predominantly probing of the epidermal cells, which favors the acquisition of virus particles on the stylet (mouthparts) for onward transmission to the next host. On Type 2 hosts CMV infection does not induce resistance to prolonged feeding. Aphids acquire nutrients from the host vascular bundle by deeper probing with their stylets, and are able to settle and reproduce. Migration of aphids (and onward virus transmission) away from Type 2 hosts is less rapid than from Type 1 hosts but Type 2 hosts provide an environment that allows aphid populations to recover and to survive challenging conditions (for example drought, cold).</p

The Fny2a protein induces anti-aphid resistance in transgenic plants.

<p>Growth rate of individual aphids feeding on transgenic Arabidopsis plants expressing one or two Fny-CMV proteins and non-transgenic (NT) plants, n≥24. Transgene expression was driven by the constitutive cauliflower mosaic virus 35S promoter. The CMV proteins expressed were the 1a (replicase component), 2a (replicase component), 2b (viral suppressor of RNA silencing), movement protein (MP), and coat protein (CP). Lines indicated by 2b/1a are double transgenic harboring <i>1a</i> and <i>2b</i> transgenes. The <i>2b</i>-transgenic lines 2.30F and 3.13F were constructed by Lewsey et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083066#B31" target="_blank">31</a>]. Different letters (a-c) are assigned to statistically different results (ANOVA with post-hoc Tukey’s tests, <i>P</i><0.05). Error bars represent standard error of the mean.</p

Fny-CMV infection of Arabidopsis induces resistance to aphids by inducing accumulation of 4-methoxy-indol3yl-methylglucosinolate (4MI3M).

<p>(A) High performance liquid chromatography analysis showed that Fny-CMV infection induced accumulation of the anti-feedant glucosinolate, 4MI3M. (B) Fny-CMV infection was unable to induce resistance in either of two independent <i>cyp81f2</i> mutant lines compromised in 4MI3M biosynthesis contrary to wild-type (WT) plants, n≥24. Error bars represent standard error of the mean. Asterisks indicate significant differences [Student’s <i>t</i>-test in (A), ANOVA with post-hoc Tukey’s tests in (B)]: *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. Non-significant: ns.</p

Fny-CMV infection of Arabidopsis induced genes that are also activated during PAMP- and effector-triggered immunity.

<p>(A) Area-proportional Venn diagram showing overlap between genes activated during PAMP- and effector-triggered immunity. Genes up-regulated by Fny-CMV infection were compared with previously published microarray data showing genes commonly induced by three PAMPs (flg22, elf26, and chitin) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083066#B84" target="_blank">84</a>], and genes induced by the <i>Pseudomonas syringae</i> effector AvrRPS4 in RPS4 Col-0 Arabidopsis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083066#B85" target="_blank">85</a>]. Venn diagram was drawn based on an image generated using a free online program (<a href="http://bioinforx.com/free/bxarrays/venndiagram.php" target="_blank"><u>http://bioinforx.com/free/bxarrays/venndiagram.php</u></a>). (B) Accumulation of the defense-related transcripts MPK3, FRK1 and <i>CYP81F2</i> was induced following infection with Fny-CMV as measured by RT-Q-PCR. Error bars represent standard error of the mean. Asterisks indicate significant differences (Student’s <i>t</i>-test): *, <i>P</i><0.05.</p