Aphid-borne Viral Spread Is Enhanced by Virus-induced ...CMVD2b-infected plants, and mock-inoculated...

Aphid-borne Viral Spread Is Enhanced by Virus-inducedAccumulation of Plant Reactive Oxygen Species1

Huijuan Guo,a Liyuan Gu,a Fanqi Liu,b Fajun Chen,c Feng Ge,a,c and Yucheng Suna,2,3

aState Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology,Chinese Academy of Sciences, Beijing 100101, ChinabDepartment of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095,ChinacUniversity of Chinese Academy of Sciences, Beijing, China

ORCID ID: 0000-0003-2353-0218 (Y.S.).

Most known plant viruses are spread from plant to plant by insect vectors. There is strong evidence that nonpersistentlytransmitted viruses manipulate the release of plant volatiles to attract insect vectors, thereby promoting virus spread. Themechanisms whereby aphid settling and feeding is altered on plants infected with these viruses, however, are unclear. Here weemployed loss-of-function mutations in cucumber mosaic virus (CMV) and one of its host plants, tobacco (Nicotiana tabacum), toelucidate such mechanisms. We show that, relative to a CMVD2b strain with a deletion of the viral suppressor of RNAi 2bprotein in CMV, plants infected with wild-type CMV produce higher concentrations of the reactive oxygen species (ROS) H2O2in plant tissues. Aphids on wild-type CMV-infected plants engage in shorter probes, less phloem feeding, and exhibit otherchanges, as detected by electrical penetration graphing technology, relative to CMVD2b-infected plants. Therefore, the frequencyof virus acquisition and the virus load per aphid were greater on CMV-infected plants than on CMVD2b-infected plants. Aphidsalso moved away from initial feeding sites more frequently on wild-type CMV infected versus CMVD2b-infected plants. The roleof H2O2 in eliciting these effects on aphids was corroborated using healthy plants infused with H2O2. Finally, H2O2 levels werenot elevated, and aphid behavior was unchanged, on CMV-infected RbohD-silenced tobacco plants, which are deficient in theinduction of ROS production. These results suggest that CMV uses its viral suppressor of RNAi protein to increase plant ROSlevels, thereby enhancing its acquisition and transmission by vector insects.

Most known plant viruses are spread from plant toplant by insect vectors. Most of these vectors are he-mipterans, a group of phloem-feeding insects such asaphids, planthoppers, and whiteflies (Antolinez et al.,2017). In plant-vector-virus interactions, the vector in-sects are the only component that can move freely inspace. Several studies have found that viruses can al-ter plant defensive signaling and modify the volatiles

released from the plant in ways that attract or repelvector insects (Mauck et al., 2010, 2014; Li et al., 2014;Wu et al., 2017). Currently, there is still a lack of vector-resistant plant varieties to control vector-transmittedviruses in agriculture, which will likely be used in thefuture for virus disease management (Bragard et al.,2013).The stylet is not only specialized for the extraction of

phloem sap, but also serves as a site where virus par-ticles can attach and be retained (Webster et al., 2017).Stylet positioning and feeding activities have importantconsequences for acquisition and transmission of virusby insect vectors (Mart et al., 1997; Jiang et al., 2000).Persistently transmitted viruses (PTVs) typically benefitfrom sustained feeding in the phloem, because suchfeeding causes a relatively large number of virus par-ticles to be translocated into the gut lumen and to becirculated within the insect (Weintraub and Beanland,2006; Hogenhout et al., 2008). Nonpersistently trans-mitted viruses (NPTVs), in contrast, benefit when insectvectors frequently change the feeding location or eventhe host plant, because these changes increase theprobability of virus acquisition and transmission(Martin et al., 1997). When an insect vector probes aplant’s epidermis/mesophyll or feeds on its phloem,the host plant reacts with physical and biochemicaldefenses, which can be altered in different ways by

1This project was supported by the National Key R&D Program ofChina (no. 2017YFD0200400) and Strategic Priority Research Pro-gram of the Chinese Academy of Sciences (no. XDB11050400), theNational Natural Science Foundation of China (nos. 31500332 and31770452), and the Youth Innovation Promotion Association of theChinese Academy of Sciences (no. 2017112).

2Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:[email protected] (Yucheng Sun).

H.G. and Y.S. conceived the original screening and research plans;H.G. and Y.S. supervised the experiments; L.G. and F.L. performedmost of the experiments; F.C. provided technical assistance to F.L.;H.G. designed the experiments and analyzed the data; H.G. and Y.S.conceived the project and wrote the article with contributions of allthe authors; F.G. supervised and complemented the writing.

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PTVs and NPTVs (Mauck et al., 2016). The PTV Tomatoyellow leaf curl China virus, for example, can directlysuppress jasmonic acid signaling and accordinglydown-regulate terpenoid synthesis by tobacco (Ni-cotiana tabacum), which improves phloem-feeding bywhitefly vectors and consequently increases acquisi-tion of the PTV (Zhang et al., 2012; Luan et al., 2013; Liet al., 2014). In contrast, the molecular mechanisms bywhich NPTVs alter host defenses to alter the feedingbehavior of insect vectors are less comprehensivelystudied.

Aphids, which are the most well-studied vectors ofplant viruses, transmit over 300 plant viruses, andmostare NPTVs (Ng and Perry, 2004; Hooks and Fereres,2006). Recent fluorescence evidence showed that thebinding site of one NPTV Cauliflower mosaic virus is lo-calized in the aphid stylet tip (Uzest et al., 2010). Thesetips, which are termed “acrostyles,” interact with cap-sid proteins or with virion-encoded accessory proteins(i.e., P2 in Cauliflower mosaic virus) of NPTVs (Uzestet al., 2010; Webster et al., 2018). During a brief periodof probing epidermal andmesophyll cells, aphid styletscan access cell contents including virions (Websteret al., 2017). Once the stylets have penetrated beyondthe epidermis and mesophyll tissues into the phloemfor sustainable passive feeding, NPTV acquisition andtransmission rates decline sharply (Nault, 1997). Aphidsgenerally probe more frequently, and for longer dura-tions, when there are stronger plant defenses in theepidermis and mesophyll tissues, i.e., when they con-front high levels of reactive oxygen species (ROS), ac-tivation of phytohormone-mediated resistance, crosslinking of the cell wall, and deposition of callose(Jaouannet et al., 2014). These plant defenses andtheir associated molecular signaling can be modifiedby some viral-encoded proteins (Westwood et al.,2014; Casteel et al., 2015; Groen et al., 2017). For ex-ample, the 2b protein, which is encoded by the NPTVcucumber mosaic virus (CMV), is a well-studiedsuppressor of host RNAi (VSR) that can target thehost’s RNA-binding protein ARGONAUTE1 (AGO1)(Zhang et al., 2006). AGO1 is required for a numberof pathogen-associated molecular pattern-triggered im-munity responses including callose deposition (Li et al.,2010).

CMV, which as noted earlier is an NPTV, is widelydistributed and is transmittedmainly by aphids (Shintakuand Palukaitis, 1990). Successful CMV infection leadsto systemic symptoms such as mosaic, chlorotic andnecrotic patterns, mottling, leaf rolling, and develop-mental abnormalities by negatively affecting physiologi-cal processes such as photosynthesis and respiration incultivated plants (Song et al., 2009). Plant photosyntheticelectron flux to carbon reduction, and respiratory electrontransport via both complex I and complex II significantlydecreased after virus infection (Díaz-Vivancos et al.,2008), whichwere accompanied by a general increase inthe activities of superoxide dismutase and ascorbate-glutathione cycle enzymes followed by an increasedH2O2 accumulation in chloroplasts and mitochondria

(Song et al., 2009; Lei et al., 2016). Furthermore, the 2bprotein of Lily-strain CMV directly interacts withcatalase (CAT3) and alters the cellular location of CAT3,a key enzyme in the breakdown of the major ROShydrogen peroxide (H2O2), in Arabidopsis (Arabidopsisthaliana). The interaction between 2b and CAT caused adecrease in catalase activity, which caused H2O2 gen-eration and induced necrosis in leaves (Inaba et al.,2011). ROS bursts cause browning of leaf cells bymodifying phenol metabolism and lignin formationand polymerization (Laitinen et al., 2017; Rasoolet al., 2017), which presumably makes it more diffi-cult for aphids to reach the sieve elements. In thisstudy, we tested the hypothesis that CMV uses itsVSR to induce ROS, which impairs phloem feedingand increases probing and movement by the aphidMyzus persicae, thereby enhancing virus acquisitionand transmission.

RESULTS

CMV infection induces plant ROS production

The production of H2O2 and other ROS is a commondefense reaction of plants against invading pathogens(Hirt, 2016). In this study, 3,39-diaminobenzidine (DAB)staining indicated that H2O2 accumulated in tobaccoplants that had been infected by CMV for two weeksbut not in CMVD2b-infected plants or mock-inoculatedplants (Fig. 1A). As indicated by trypan blue staining,cell death was greater in CMV-infected plants than inCMVD2b-infected plants or mock-inoculated plants(Fig. 1B). Because the production of ROS disturbs theelectron transport chains in PSII, the maximum quan-tum yields of PSII were lower in CMV-infected plantsthan in CMVD2b-infected plants or mock-inoculatedplants (Fig. 1C). We further determined the H2O2 con-centration and then the NADPH oxidase activity, a keyenzyme involved in ROS production. The CMV-infected plants had a higher H2O2 concentration andNADPH oxidase activity than CMVD2b-infectedplants or mock-inoculated plants (Fig. 1, D and E).This indicated that CMV infection can induce ROSproduction in tobacco and that the VSR 2b protein wasresponsible for generating the ROS signals in plants.Fluorescent probes indicated that, at two weeks afterinoculation, H2O2 was abundant in both the intracel-lular and intercellular spaces of mesophyll cells ofCMV-infected plants but not of CMVD2b-infectedplants (Fig. 1F).

CMV infection increases intracellular probing by aphids

To determine the effects of CMV infection and VSRon aphid feeding behavior, CMV-infected plants,CMVD2b-infected plants, and mock-inoculated plantswere used to assess aphid feeding using electrical pene-tration graphing (EPG) (Fig. 2A). At 4 h after landingon host plants, 76.7% of the aphids on CMV-infected

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Plant ROS enhance virus spread by aphids

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plants had moved to new leaves, whereas only 16.7% onmock-inoculated plants and only 20.0% on CMVD2b-infected plants had moved to new leaves (Fig. 2B).Aphids on CMV-infected plants spent more time(2.09 6 0.05 h) reaching the phloem than aphids onmock-inoculated or CMVD2b-infected plants (Fig. 2C).Before reaching the phloem for the first time, aphidsassociated with CMV-infected plants exhibited moreintracellular probing than aphids on mock-inoculatedor CMVD2b-infected plants (Fig. 2D). Before shifting toa new feeding site, which is the average duration beforethe second nonpenetration (NP) phase, aphids onCMV-infected plants spent less time (0.476 0.02 h) thanthose on mock-inoculated (4.2 6 0.11 h) or CMVD2b-infected (3.9 6 0.03 h) plants (Fig. 2E). These resultssuggested that aphids on CMV-infected plants spentmore time in the pathway phase, waveform C, than in

phloem feeding in the first 2 h after placement on hostplants. We then analyzed the aphid feeding activities inthe first and second 2-h period (0 h to 2 h and 2 h to 4 h)after aphids were placed on host plants. In the 0-h to 2-hperiod, aphids on CMV-infected plant spent more timein the pathway phase but less time in the phloemfeeding phase than those on CMVD2b-infected plantsor mock-inoculated plants (Fig. 2F). In the 2-h to 4-hperiod, however, aphids on CMV-infected plantsspent more time in the nonprobing phase and less timein both the intracellular pathway and phloem feedingphase than those on CMVD2b-infected plant or mock-inoculated plants (Fig. 2G). In the 0-h to 2-h period,probe numbers by aphids were greater on CMV-infected plants than on CMVD2b-infected plants ormock-inoculated plants (Fig. 2F). These results sug-gested that aphids on CMV-infected plants spent more

Figure 1. CMV infection induces a stronger ROSburst than CMVD2b infection or mock-inoculation inwild-type plants. Representative images and data arefrom leaves two weeks after CMV infection, CMVD2binfection, or mock inoculation. A to C, Representativeimages of leaves after (A) DAB staining (H2O2 indi-cator); B, trypan blue staining (cell death indicator);C, Chlorophyll fluorescence images for maximumquantumyield of PSII (Fv/Fm) (leaf senescence or stressindicator). D, H2O2 production and (E) NADPH oxi-dase activity; each value is the mean (SE) of 10 repli-cates. Means with different letters are significantlydifferent as described by Tukey’s multiple range testanalysis (P , 0.05). F, Subcellular localization offluorescent probes, H2DCF-DA; images of the probe(green) and chlorophyll (red) fluorescence emissionare shown together with overlay images. Scale bar,20 mm.

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time in the pathway phase (in terms of C waveform)during the first 2 h of feeding and spent more timeengaged in nonprobing activities during the second 2 hof feeding.

Application of exogenous H2O2 to host plants increasesintracellular probing time and movement of aphids

To further confirm the effects of host plant ROS onaphid feeding behavior, we determined the feedingactivities of aphids on plants that had been infiltratedwith 20 mM H2O2. Four hours after placement on hostplants, the percentage of aphids that moved to newleaves was 56.6% on plants with H2O2 infiltration and26.6% on plants without H2O2 infiltration (Fig. 3A).EPGs showed that exogenous H2O2 infiltration en-hanced the pathway phase and intracellular probingnumbers but dramatically decreased the phloem feeding

time (Fig. 3, C, D, and F). Furthermore, aphids spentmore time reaching the phloem on H2O2-infiltratedplants than on noninfiltrated plants (Fig. 3G). Theseresults suggested that the accumulation of ROS in hosttissues is unfavorable for aphid feeding in that it in-creases the duration of the penetration phase but re-duces the phloem feeding time.

VSR enhances virus acquisition by aphids

Aphids associated with CMV-infected wild-typeplants acquired more virus than CMVD2b-infectedplants during the first 2 h of feeding (Fig. 4, A and B).A quantity of 63.3% aphids associated with CMV-infected wild-type plants acquired virions while only26.7% aphids associated with CMVD2b-infected plantsacquired virions during the first 2 h of feeding (Fig. 4C).The CMV copy numbers acquired by aphids were

Figure 2. CMV infection increases thefrequency of intracellular probing and in-creases changes in feeding location byaphids. A, Schematic representation of thestudy. Movement and feeding behavior ofaphids on CMV-infected, CMVD2b-infec-ted, andmock-inoculatedwild-type plantswere detected during a 4-h feeding periodby using EPG technology. Waveform pat-terns were scored according to previouslydescribed categories: NP; pooled pathwayphase activities in intercellular space ofepidermis and mesophyll cells (waveformC); short intracellular punctures in epi-dermis and mesophyll cells (pd); salivarysecretion into phloem sieve elements (E1);and phloem ingestion (E2). B, The per-centage of aphids that moved to newleaves during a 4-h feeding period onCMV-infected, CMVD2b-infected, andmock-inoculated wild-type plants. C,The time required for the stylet to firstreach the phloem. D, Number of probesbefore phloem ingestion. E, Time to firstchange in feeding site. F and G, Totaltimes spent in each waveform (includingNP, “C,” E1, and E2) as well as pd numbersduring (F) the first 2 h of feeding and during(G) the second 2 h of feeding. Each value isthe mean (6SE) of 30 replicates. Meanswith different letters are significantly dif-ferent as described by Tukey’s multiplerange test analysis (P , 0.05).





positively correlated with the intracellular pathwaytime (r2 = 0.533, P , 0.001) and the total probingnumbers (r2 = 0.604, P , 0.001; Figure 4, D and E).During the first five probes after aphids were placed onplants, CMV copy number per aphid and the percent-age of aphids with CMV did not significantly differ onCMV-infected and CMVD2b-infected plants (Fig. 4, Fand G).

Effects of RbohD-silenced plants on feeding behavior andvirus acquisition of aphids

ROS production during plant pathogen interactionsdepends on plasma membrane (PM)-located respiratoryburst oxidase homolog (Rboh) enzymes (Groom et al.,1996; Desikan et al., 1998; Amicucci et al., 1999; Torresand Dangl, 2005). In N. tabacum, RbohD is required forthe induction of ROS production (Noirot et al., 2014). Tofurther verify the effect of ROS on virus acquisition byaphids, RbohD-silenced plants (irRbodD plants) wereused to conduct loss-of-function experiments; the irR-bodD plants were artificially deficient in ROS accu-mulation. We found that for both CMV-infected andCMVD2b-infected plants, the copy numbers and therelative transcript level of 2b did not differ betweenirRbohD plants and wild-type plants (Fig. 5, A and B).Compared with CMV-infected wild-type plants, bothCMV-infected and CMVD2b-infected irRbodD plantshad lower levels of H2O2 in the intracellular and inter-cellular spaces of mesophyll cells and had lowerNADPH oxidase activity (Fig. 5, C–E). The results in-dicated that CMV infection failed to trigger ROS accu-mulation in irRbodD plants and that the reduction inROS accumulation in irRbodD plants did not affectCMV replication. This indicated that the accumulationof ROS in CMV-infected wild-type plants did not re-duce CMV infection.We also determined the feeding behavior of aphids

on CMV-infected and CMVD2b-infected irRbohD plants.

The total time spent in the pathway phase, the totalintracellular short probing numbers, and the phloemingestion phase did not significantly differ for aphidson CMV-infected versus CMVD2b-infected irRbohDplants (Fig. 6, A–C). Similarly, the percentage ofaphids that moved from one leaf to another and theCMV accumulation in aphids did not significantlydiffer on CMV-infected versus CMVD2b-infected irR-bohD plants (Fig. 6, D and E). CMV accumulation waslower in aphids on CMV-infected irRbohD plants thanon CMV-infected wild-type plants (Figs. 4B and 6E).These results suggested that the plant ROS signalingpathway is necessary for the CMV-induced enhance-ment of intracellular probing and virus acquisition byaphids.

Expression of CMV 2b protein in plants affects ROSaccumulation and aphid feeding behavior

To determine the effects of the CMV 2b protein onROS accumulation and aphid feeding behavior andvirus acquisition, tobacco plants expressing the CMV2b protein were obtained by agroinfiltration. For thispurpose, the coding sequence of 2bwas cloned into thepotato virus X (PVX)-based plant binary expressionvector (p35S-P2C2S) to form pPVX-2b. pPVX-2b wasagroinfiltrated into wild-type tobacco leaves to ob-tain PVX2b plants that express 2b protein transiently.Relative to PVX plants, which were agroinfiltratedwith the empty vector PVX, the PVX2b plants hadlower maximum quantum yields of PSII (Fig. 7A),increased cell death (Fig. 7B), increased H2O2 pro-duction (Fig. 7C), and increased NADPH oxidaseactivity (Fig. 7D). These results suggested that the 2bprotein of CMV can directly induce ROS productionin tobacco.During the first 2 h after placement on plants, aphids

on PVX2b plants spent more time in the intercellularpathway phase and less time in phloem feeding than

Figure 3. Exogenous H2O2 treatment increases thechanges in feeding location and increases the fre-quency of intracellular probing by aphids. All datawere obtained over a 2-h feeding period. A, Per-centage of aphids that moved to new leaves whenplaced on plants with and without exogenousH2O2 treatment. B, D to F, Total time spent in B, NP,(D) pathway phase activities (waveform C) in in-tercellular space of epidermis and mesophyll cells(B), (E) salivation into phloem sieve elements (E1),and (F) phloem ingestion (E2). F, Short probenumbers (pd). G, Time required to reach the phloemfor the first time. Each value is the mean (6SE) of 30replicates. Means with different letters are signifi-cantly different as described by independent t testanalysis (P, 0.05).


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those on PVX plants (Fig. 7, E and H). Furthermore,intracellular probing numbers were higher on PVX2bplants than on PVX plants (Fig. 7F).

The effects of 2b protein expression by plants on virusacquisition and movement of aphids

CMV copy number and 2b transcript level werelower in CMVD2b-infected than in CMV-infected PVXplants at 6 days, 8 days, and 10 days after inoculation(Fig. 7, I and J). In contrast to these data, CMV copynumber and 2b transcript level were similar inCMVD2b-infected PVX plant and CMV-infected PVX2bplants. This suggested that the expression of the 2bprotein can restore the ability of CMVD2b to infectplants.

At 6 days, 8 days, and 10 days after inoculation,the percentage of aphids that changed feeding sitesfrom one leaf to another during a 4-h feeding periodwas much higher on CMVD2b-infected PVX2b plants

(50%, 53.3%, and 60%, respectively) than on CMVD2b-infected PVX plants (16.7%, 13.3%, and 20%, respec-tively) (Fig. 7K). The percentage that changed feedingsites was relatively similar on CMV-infected PVXplants versus CMVD2b-infected PVX2b plants (Fig. 7K).

At 6 days, 8 days, and 10 days after inoculation, thepercentage of aphids that acquired CMV particles duringa 2-h feeding period was much higher on CMVD2b-infected PVX2b plants than on CMVD2b-infected PVXplants (Fig. 7L). The percentage that acquired CMV par-ticles was relatively similar on CMV-infected PVX plantsversus CMVD2b-infected PVX2b plants. The trends forcopy number per plant (Fig. 7I) were similar to those forvirus acquisition per aphid (Fig. 7L).

DISCUSSION

According to the “pull and push” hypothesis, plantsinfected by NPTVs initially attract vectors and stimu-late aphids to ingest cell contents to enhance virion

Figure 4. CMV-induced ROS facilitates CMV acqui-sition via aphid intracellular probing in epidermis andmesophyll cells. A, Schematic representation of theexperiment. After plants had been infected by CMVand CMVD2b for two weeks, the feeding activities ofaphids were monitored on these plants by EPG; inaddition, CMV copy number and the percentage ofaphids that acquired CMV was determined for 30aphids that fed for 2 h and for another 30 aphids thathad penetrated the host five times (five probes). B,CMV copy number per aphid after 2 h of feeding; thenumber of aphids that acquired CMV relative to totalaphids is shown at the top of each column. Each dotrepresents an aphid. C, Percentage of aphids thatacquired CMV after 2 h of feeding. For B and C, eachvalue is the mean (6SE) of 30 replicates. Means withdifferent letters are significantly different as describedby independent t test analysis (P , 0.05). D, Corre-lation between short probe numbers during thepathway phase and CMVcopy number per aphid after2 h of feeding on CMV-infected and CMVD2b-infected plants. E, Correlation between the time ofthe total pathway phase time and CMV copy numberper aphid after 2 h of feeding on CMV-infected andCMVD2b-infected plants. F, CMV copy number peraphid after five probes; the number of aphids thatacquired CMV relative to total aphids is shown at thetop of each column. Each dot represents an aphid. G,Percentage of aphids that acquired CMV after fiveprobes. In F and G, values are means (6SE) of 30replicate aphids; means with different letters are sig-nificantly different (P , 0.05) as described by inde-pendent t test analysis. NS, not significant.





acquisition, but then cause vectors to move to healthyplants so as to enhance transmission (Martin et al., 1997;Carmo-Sousa et al., 2014). The results of this studysuggest that the CMV VSR 2b protein triggers an in-crease in H2O2 production in tobacco and that this in-crease in H2O2 altersM. persicae probing frequency andmovement in ways that enhance virion acquisition andsubsequent aphid dispersal.Although differing in the duration and specificity of

their interactions with insect vectors, most PTVs andNPTVs rely on insect vectors for plant-to-plant spread(Ng and Falk, 2006; Hogenhout et al., 2008). Given thateffective transmission is critical to the fitness of vector-borne viruses, wemight expect that these pathogens arefrequently under selection to change host phenotypesand effects on vectors that facilitate transmission(Mauck et al., 2016). Previous studies found that PTVstend to enhance host palatability and encourage vectorsettling and feeding, while NPTVs often exhibit neutralor negative effects on the palatability of infected hostsfor insect vectors (Castle and Berger, 1993; Eigenbrodeet al., 2002; Zhang et al., 2012). For instance, CMV en-hances aphid attraction to cucurbit hosts by elevatingvolatile emissions of infected plants while also re-ducing plant palatability by altering nutrient cues,which encourages aphid dispersal after probing be-havior (Mauck et al., 2010, 2014). In this study, wefurther confirmed that the VSR 2b protein of the SDstrain of CMV could directly trigger plant H2O2 pro-duction, an aphid feeding deterrent that stimulatesdispersal after probing. The effect of 2b protein on hostplant resistance varies depending on virus and the hostspecies. For instance, the 2b protein of the Fny strain ofCMV diminished resistance to aphid infestation inCMV-infected tobacco plants by suppressing the jas-monic acid signaling pathway but increased resistanceagainst aphids in CMV-infected Arabidopsis plants byactivating biosynthesis of the aphid feeding deterrent4-methoxy-indol-3-yl-methylglucosinolate (Ziebell et al.,2011; Westwood et al., 2013; Tungadi et al., 2017). Fur-ther sequence alignment of the 2b protein in differentCMV strains suggested that some sequence variationsin the C-terminal of the 2b protein may contribute to itscontrasting modification of plant defensive signalingpathways (Duan et al., 2012), because it has beendemonstrated that the physical interaction of the 2bprotein with host plant proteins such as AGO1 andCAT3 requires the C-terminal region (Inaba et al., 2011;Zhang et al., 2012).Rapid generation of ROS is one of the earliest cellular

responses of plants to various biotic stresses includingplant viruses (Hirt, 2016). Because ROS cause host celldeath, ROS have long been considered to be harmfulby-products of virus infection (Yoshioka et al., 2003;Choi et al., 2007). Recent studies have found, how-ever, that virus proteins can interact with plant hostproteins to either increase or suppress ROS produc-tion by the host plant and thereby facilitate virus in-fection. For example, the replication protein p27 ofred clover necrotic mosaic virus (RCNMV) recruits the

Figure 5. Silencing of RbohD in plants suppresses the induction of ROSby the 2b protein of CMV. A, Copy numbers of CMV and B, relativeexpression of the CMV 2b gene in wild-type and irRbohD plantsinfected by CMVor CMVD2b for two weeks before aphid infestation. C,H2O2 production and D, NADPH oxidase activity in wild-type andirRbohD plants infected by CMVor CMVD2b without aphid infestation.In A to D, each value is the mean (6SE) of six replicates and means withdifferent letters are significantly different (P , 0.05) as described byTukey’s multiple range test analysis. E, Subcellular localization of flu-orescent probes in CMV-infected and CMVD2b-infected leaves in wild-type and irRbohD plants two weeks after inoculation. Separate imagesof the H2DCF-DA probe (green), chlorophyll (red) fluorescence emis-sion, and merged images are shown. Lg, log to base 10; WT, wild type.Scale bar, 20 mm.


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ROS-generating enzyme RbohB of N. benthamiana fromthe Golgi to sites of viral RNA replication, which in-duces ROS production so as to enhance RCNMV rep-lication (Hyodo et al., 2017). The VSR gb protein ofbarley stripe mosaic virus, in contrast, interacts withglycolate oxidase and inhibits peroxisomal ROS pro-duction to facilitate virus infection (Yang et al., 2017).Thus, the generation of ROS may be a common re-sponse to virus infection, but whether ROS enhances orsuppresses virus infection appears to be species-specific. In this study, the VSR 2b protein of CMV in-duced ROS production inN. tabacum, but the increase inROS production did not directly affect CMV infectionand replication because CMV copy numbers did notsignificantly differ between CMV-infected irRhoBD andwild-type plants in the absence of aphids (Fig. 5). Thisindicates that the virus-induced increase in ROS gener-ation did not directly promote virus spread in hostplants. Because RCNMV is soil-transmitted and barleystripe mosaic virus is transmitted through plant-to-plantcontact, pollen and seed (neither of which rely on insectvectors), while CMV is largely transmitted by insectvectors, it seemedpossible at the outset of this study that,in the case of CMV, ROSmight enhance virus replicationand spread via its effects on vector behavior.

In a compatible plant–aphid interaction, aphids canrapidly locate the sieve elements after landing and canfeed on the phloem for long periods without causingsignificant damage to epidermis and mesophyll cells(Garzo et al., 2019). This behavior limits the activationof plant defenses against aphids but would be unfa-vorable for NPTV acquisition and transmission by

aphids. In nature, CMV has a very broad host range,infecting more than 1200 plant species across more than100 families, and the CMV virions are transmitted in astylet-borne manner by more than 80 species of aphid(Scholthof et al., 2011). For many CMV host plants, theyare nonhost of some aphid vector species but still couldbe infected by the aphid-borne virus because of anaphid’s probing. Therefore, in many cases, the non-colonizing aphids that probe and move through aphidnonhost plants are primarily responsible for the spreadof NPTVs (Hooks and Fereres, 2006; Ng and Falk, 2006),suggesting that the selection of host plants may favorNPTVs that canmodify aphid behavior so as to increasevirus acquisition and transmission. In this study, the 2bprotein of CMV induced ROS generation in both theintracellular and intercellular spaces of plant mesophyllcells (Fig. 1F). The production of ROS could promotecell wall thickening and callose deposition in the apo-plast of host plants, which may prevent aphid phloemfeeding (Gao et al., 2008; Rasool et al., 2017). We furtherconfirmed that treating host plants with exogenousH2O2 or causing plants to overexpress the 2b protein ofCMV resulted in an increased number of intracellularprobes during the 2-h period after aphids were placedon host plants. If ROS generation in the plant apoplastwas impaired by RNAi of RbohD, the short probenumbers were reduced. The VSR 2b protein of CMValso increases the levels of intracellular and intercellularROS in the host plant, which increases aphid penetra-tion into epidermal andmesophyll cells where the virusparticles can bind to the aphid stylet tips instead offlowing into the aphid gut.

Figure 6. Silencing of RbohD in plants increasesfeeding efficiency but decreases virus acquisition byaphids. The data in A to Cwere obtained from aphidsthat fed for 2 h on mock-inoculated, CMV-infected,and CMVD2b-infected irRbohD plants. A, Total timespent in the pathway phase (waveform C); B, shortprobe numbers (pd); C, total time spent in phloemingestion (E2); and D, percentage of aphids move tonew leaves during 4 h of feeding on mock-inoculated,CMV-infected, and CMVD2b-infected irRbohD plants.E, Average CMV copy number per aphid on irRbohDplants infected by CMV or CMVD2b; the number ofaphids that acquired CMV relative to total aphids isshown at the top of each column. Each dot representsan aphid. In each panel, each value is themean (6SE) of30 replicates. Means with different letters are signifi-cantly different (P , 0.05) as described by Tukey’smultiple range test analysis.





Figure 7. Overexpression of 2b protein in plant leaves induces ROS production and increases aphid penetration and virus ac-quisition. Representative leaf images of (A) chlorophyll fluorescence, i.e., maximum quantum yield of PSII (Fv/Fm) (leaf senes-cence or stress indicator) and (B) trypan blue staining (cell death indicator) 10 days after transient expression of 2b protein inleaveswith the PVX vector. C, H2O2 production and (D)NADPHoxidase activity of PVX-2b plants and empty-vector (PVX) plants.Each value is the mean (6SE) of six replicates. Panels E to H concernM. persicae feeding activities during 2 h onmock-inoculated,CMV-infected, and CMVD2b-infected wild-type plants (data were collected twoweeks after inoculation): total time spent in E thepathway phase (waveform C), F intercellular puncture (pd), (G) salivation, and (H) the phloem ingestion phase. For E to H, each


Guo et al.



Because viruses have a limited coding capacity, theyhave evolved some multifunctional proteins that modifyhost factors so as to favor infection (Pumplin andVoinnet, 2013). The VSR 2b protein of CMV, in addi-tion to inducing ROS production, directly interacts withthe jasmonate (JA) ZIM-domain protein and therebysuppresses JA-responsive genes such as the Helix-Loop-Helix transcription factors MYC2, MYC3, andMYC4 (Wu et al., 2017). The suppression of MYCsfurther represses the biosynthesis of plant volatiles andenhances the odor-dependent attractiveness to aphidvectors. The 2b protein not only down-regulates MYCsto increase aphid attraction but, according to our re-sults, also increases ROS to enhance virus acquisitionand transmission by aphids. This indicates that theplant-mediated effects of the 2b protein on insect be-havior are consistent with the pull–push strategycharacteristic of some other NPTVs. Further work isrequired, however, to determine how the 2b proteincoordinates plant ROS signaling and the JA signalingpathway in response to virus infection and insect vectorinfestation.

In summary, this study has generated several im-portant findings. Firstly, CMV used its VSR to increasethe production of plant ROS, which did not directlyinhibit CMV infection in tobacco. Secondly, the 2b-induced ROS was localized in both the intercellularand intracellular space of mesophyll cells, whichdelayed phloem feeding by aphids, increased the pen-etration frequency in mesophyll cells, and increased thefrequency at which aphids moved to new leaves.Overall, these results confirm that the VSR of a stylet-borne virus triggers plant defensive signaling directlyand modifies vector feeding behavior to increase vi-rus acquisition (by increasing contact between thevirus and the vector stylet) and aphid movementamong host leaves. The increase in virus acquisitionand in movement by aphids presumably increasesvirus transmission.

MATERIALS AND METHODS

Plant, vector insect, and virus

Nicotiana tabacum (genotype: W38) plants were used for all experiments andtransformations. Seedlings were grown under a 16-h (23°C)/8-h (21°C) light/dark photoperiod with 400 mmol m22 s21 of active radiation supplied duringthe light period. The tobacco-feeding strain of the aphid Myzus persicae wascollected from tobacco fields. The nymphal instars from the same partheno-genetic female were reared on N. tabacum seedlings in photoclimatic chambersformore than 10 generations before experiments were initiated. The SD strain ofCMV was originally isolated from tobacco in the Shandong Province of China

and was propagated in N. tabacum. Its 2b gene-deletion mutant, CMVD2b, waskindly provided by Professor Huishan Guo from the Institute of Microbiology,Chinese Academy of Sciences (Fang et al., 2016).

Generation of stably RbohD-silenced N. tabacum plants

We generated stably RbohD-silenced plants through Agrobacterium tumefaciens-mediated transformation as described by Krügel et al. (2002). In brief, the RbohDfragment, amplified with the forward primer 59-ATTGGTGGGTCTTGGAAT-39and the reverse primer 59-AACGAGCATCACCTTCTTCA-39was inserted intothe pRESC5 transformation vector in an inverted-repeat orientation. Seedlinghypocotyls were cut into 3-mmpieceswith a scalpel that had been dipped in theAgrobacterium suspension. After callus induction and selection, light greenshoots began to develop. The callus with shoots was subcultured every threeweeks until plantlets formed. Plantlets were subcultured onto a rooting me-dium every three weeks until roots appeared, after which plants were carefullyremoved from the gel and planted in soil. When the plants produced seeds,the seeds were collected, screened with hygromycin, and used for furtherexperimentation.

PVX agroinfection and agroinfiltration assays of 2b protein

For assays concerning the overexpression of 2b protein, the full-length cDNAclone of the potato virus X (PVX) vector, pP2C2S, kindly provided byDr. DavidBaulcombe (The Sainsbury Laboratory, John Innes Centre, Norwich, UK), wasused to construct recombinant PVX-2b clones. Polymerase-chain-reaction(PCR)–amplified sequences coding for SD2b were cloned into p35S-P2C2S. The35S promoter-driven recombinant PVX-2b viral vector or the PVX vector alonewere transformed into Agrobacterium EHA105 for agroinoculation (Ye et al.,2009).

CMV inoculation

After four weeks’ growth, the wild-type plants and irRbohD plants wererandomly divided into three treatment groups. These groups were used for thevarious assays and determinations described later in “Methods.”

In the first treatment group, 50 four-week-old wild-type and 50 four-week-old irRbohD N. tabacumwere inoculated with 50 mL of purified virions of CMVat a concentration of 200 mg/mL, and 50 other four-week-old wild-type and 50four-week-old irRbohD N. tabacum were inoculated with 50 mL of the purifiedvirions of CMVD2b. For mock treatment, 40 wild-type plants were inoculatedwith 50 mL of buffer (5 mM sodium borate and 0.5 mM disodium EDTA,pH = 9.0). Two weeks later, the CMV- and CMVD2b-inoculated plants wereharvested for quantification of CMV copies and 2b gene expression (10 repli-cates per treatment); plants from each treatment were harvested for histo-chemical assays of ROS accumulation levels (10 replicates per treatment). Theplants were also used for aphid feeding behavior bioassays (30 replicates pertreatment) and virus acquisition bioassays (30 replicates per treatment).

In the second treatment group, 30 four-week-oldwild-typeN. tabacum plantswere infiltrated with 100 mL of 20 mM H2O2 and another 30 four-week-old wild-typeN. tabacum plants were infiltrated with 100 mLwater. The plants were usedfor aphid feeding behavior bioassays (30 replicates per treatment).

In the third treatment group, 40 four-week-old wild-type N. tabacum plantswere agroinoculated with PVX-2b, and the PVX-2b agroinoculated plants werethen inoculated with 50 mL of purified virions of CMVD2b one week later.Another 80 plants were agroinoculated with the empty PVX vector. Half ofthese PVX agroinoculated plants were inoculated with 50 mL of purified virionsof CMV and the other half were inoculated with 50 mL of purified virions ofCMVD2b one week later. The plants from these treatment combinations wereassessed for CMV copy number, ROS level, and gene expression level at three

Figure 7. (Continued.)value is the mean of 30 replicates. Means I to L concern plants coinfiltrated with the empty vector PVX and CMVD2b, the 2bprotein-expressing vector PVX-2b and CMVD2b, and the empty-vector PVX and CMV (data were collected 6 days, 8 days, or10 days after infiltration): (I) CMV copy number per plant, (J) the relative expression of 2b protein, (K) the percentage of aphids thatmoved to new leaves, and (L) average CMV copy number per aphid. For I to L, each value is the mean of 30 replicates. Meanswithdifferent letters are significantly different (P, 0.05) as described by Tukey’s multiple range test analysis within each time point. InL, the number of aphids that acquired CMV relative to total aphid numbers is shown at the top of each column. Lg, log to base 10.Error bars indicate SEs.





time points (6 days, 8 days, and 10 days) after virus inoculation as indicated inthe “Results.” For each determination, each treatment was represented by fivereplicate plants. The plants were also used for aphid feeding behavior bioassaysand virus acquisition bioassays at three time points (6 days, 8 days, and 10 days)after virus inoculation as indicated in “Results.” Thirty replicate plants pertreatment are indicated in the descriptions of both feeding behavior bioassaysand virus acquisition bioassays.

Plant damage and histochemical assays

To visualize H2O2 accumulation, we stained the leaves with DAB. Leaves at14-d postinfection, as well as control leaves, were collected and vacuum-infiltrated with DAB solution (1 mg/mL DAB in pH 3.5 water) in a six-welltiter plate. After an overnight incubation in the same solution in darkness, theleaves were destained in 95% (w/v) ethanol until they turned clear. Imageswere then captured with a digital camera (Canon).

Trypan blue staining was used to visualize cell death. Trypan blue wasdissolved in a lactophenol solution (phenol/lactic acid/glycerol/water [1:1:1:1])at a concentration of 0.125mg/mL.Leaves preparedasdescribed in thepreviousparagraph were boiled in this staining solution for 1 min. After cooling, leafsamples were destained in 95% (w/v) ethanol and were photographed with aSZX2-ILLK microscope (Olympus).

Because ROS production can disrupt electron transport chains in PSII, ROS isnegatively correlated with maximum quantum yields of PSII. The chlorophyllfluorescence induction kinetics of predarkened leaves (20 min) were measuredusing the fluorescence imaging system FluorCam 700MF (Photon Systems In-strument) (Chi et al., 2008). The Fo (minimum fluorescence in the dark-adaptedstates) was measured under light (650 nm) with very low intensity (0.8 mMolm22 s21). To estimate the Fm (maximum fluorescence in the dark-adaptedstates), a saturating pulse of white light (2500 mMol m22 s21) was applied.The maximal photochemical efficiency of PSII was calculated from the ratio ofFv to Fm [Fv/Fm = (Fm 2 Fo)/Fm].

H2O2 production and NADPH oxidase activity were determined accordingto previous studies (Sagi and Fluhr, 2001; Lei et al., 2016). To localize H2O2 inleaves after CMV or CMVD2b infection, we vacuum-infiltrated leaves fromeach treatment with the fluorescent probe 29,79-dichlorofluorescein diacetate(H2DCF-DA), a fluorescent dye precursor for H2O2, in pH 7.4 phosphate bufferin a closed syringe for 10–30 s. The leaves were then placed in the dark for45 min. The localization of the fluorescent probe in tobacco leaves was visual-ized using a TCS SP5 laser confocal microscope (Leica Microsystems). The ex-citation energy was produced by an argon laser at 488 nm, and bands in therange 510 nm to 550 nm and 650 nm to 750 nmwere observed. A 90-nm pinholewas used. Leaf cuttings were sandwiched between two microscope coverslipsand measured. The leaf adaxial side faced the 488-nm argon laser excitation.Fluorescence emission was observed through a band at 510 nm to 550 nm fordichlorofluorescein and 650 nm to 750 nm for the red fluorescence of chloro-plasts (Lei et al., 2016). The images were analyzed using imaging systemsoftware.

Aphid feeding behavior

The feedingbehavior ofM. persicae onCMV-infected, CMVD2b-infected, anduninfected wild-type plants and on irRhobD aswell as the recombinant PVX-2band PVX plants was studied via EPG analysis (Manual-Giga-8d 17 https://www.epgsystems.eu/downloads-install-files-manuals, section Manuals). Allaphids were subjected to a 1-h preacquisition starvation period before startingwaveform recordings. Adult aphids were immobilized on ice and the aphiddorsum attached to a gold wire (2 cm long, 18.5 mm in diameter; EPG Systems)using a hand-mixed, water-based silver conductive paint glue (EPG Systems).Next, the other side of the gold wire was glued with a droplet of paint to acopper extension wire, 2 cm in length, which was inserted into the input of theEPG headstage amplifier. Another copper electrode, 10 cm long and 2 mm indiameter, was inserted into the soil of the plant container. One apterous adultM. persicae was placed on a single trifoliate leaf, and its feeding behavior wasmonitored. An eight-channel amplifier simultaneously recorded eight indi-vidual aphids on separate plants for 4 h. Waveform patterns in this study werescored according to described categories: NP; intercellular apoplastic styletpathway in which the insects show a cyclic activity of mechanical stylet pene-tration and secretion saliva (waveform C); short intracellular punctures (pd);secretion of saliva into phloem sieve elements at the beginning of the phloemphase (E1); and passive phloem sap uptake from the sieve element, i.e., phloemingestion (E2) (Tjallingii, 2006; Garzo et al., 2018). Furthermore, three EPG

parameters were considered relevant to virus acquisition by aphids: (1) time tofirst phloem ingestion (a prolonged period before phloem ingestion is thoughtto indicate that the aphid is expending time overcoming plant resistance); (2)the number of probes before the first phloem ingestion (high probing numbersbefore first phloem ingestion indicate that the aphid is spending time in intra-cellular penetration of epidermis/mesophyll tissues); and (3) the time until theend of the first bout of phloem feeding (which indicates that the aphid haswithdrawn its stylet from the phloem before changing feeding sites). The EPGanalysis was based on data collected from 30 aphids for each plant treatment.After the EPGswere recorded, the aphidswere collected, and eachwas assessedfor virus copy number. We also determined the effects of numbers of pd onvirus acquisition ability as follows: aphids were allowed to feed on CMV-infected plants and CMVD2b-infected plants, but their feeding was artificiallyinterrupted by removing the aphid from the plant at the end of the fifth drop inpotential, i.e., in the pd waveform pattern. Virus copy number in each aphidwas determined by reverse transcription PCR (see next section).

Determination of CMV copy number

The RNeasy Plant Mini Kit and RNeasy Micro Kit (Qiagen) were used toisolate total RNA fromN. tabacum leaves (50mgper sample) andM. persicae (oneaphid per sample). A TaqMan qPCR assay was used for the detection andquantification of the CMV sequences (Candresse et al., 1998). The primers of thecoat protein gene (F: 59-AAC CAG TGC TGG TCG TAA CC-39; 59-GAC CAGCTG CCA ACG TCT TA-39) generate a 165-bp DNA fragment after amplifi-cation. The TaqMan probe (59-CCC GCT CCG CTT CCT CCT CCG C-39) waslabeled with fluorescent dyes, i.e., with 6-carboxyfluoroscein on the 59-end andwith N,N,N9,N9-Tetramethyl-6-carboxyrhodamine on the 39-end. The actingene of N. tabacum and the Rpl7 gene of M. persicae were used as internal con-trols. A linear equation of CMV virus copy numbers (log transformed) versusthe Ct curve was generated. The recombinant plasmid pBI221 was constructedby the PCR product (coat protein of CMV) insertion into pGEM-T Easy vector(Promega). The standard curve of the CMV coat protein gene was obtained byusing serial 10-fold diluted plasmids (9.16 3 107 to 9.16 3 103 copies) as tem-plates. For calculation of the Y value used for the determination of viral copynumber in the tested DNA samples, the following equation was applied byusing the standard formula for the regression analysis calculation: Y =23.354X+35.27; it was possible to quantify the viral copy number in the examinedsamples. Amplifications using theMx 3500P detection system (Stratagene)wereperformed in 25-mL volumes containing 12.5 mL of Premix Ex Taq (ProbeqPCR), 2mL of template, and 1mL of each 10mM gene-specific primer as follows:95°C for 10 s, followed by 40 cycles of 10 s at 95°C and 30 s at 57°C.

Statistical analyses

PASW Statistics 18.0 (SPSS) was used for statistical analyses. One-wayANOVAswere used to analyzeH2O2 production andNADPHoxidase of plant;CMV copy numbers and CMV 2b expression of plant and aphids; and aphidfeeding behavior and percentage of aphids moving to new leaves on differenttreatments. Tukey’s multiple range test analysis was used for pairwise com-parisons of the difference between treatments for mean separation (P , 0.05).Student’s t tests were used to analyze H2O2 production andNADPH oxidase ofplant; CMV copy numbers and CMV 2b expression of plant and aphids; andaphid feeding behavior and percentage of aphids moving to new leaves on twotreatments. Linear regression analysis was used to analyze the correlation be-tween aphid feeding behavior and CMV copy numbers of aphids.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers AB008777(CMV CP), D86330.1(CMV 2b),and EF366670.1(RbohD).

Received April 16, 2018; accepted October 23, 2018; published October 31, 2018.

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Aphid-borne Viral Spread Is Enhanced by Virus-induced ...CMVD2b-infected plants, and mock-inoculated...

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