Produção Nacional
Revisado por pares
Host Plants Indirectly Influence Plant Virus Transmission by Altering Gut Cysteine Protease Activity of Aphid Vectors
2016; Elsevier BV; Volume: 16; Issue: 4 Linguagem: Inglês
10.1074/mcp.m116.063495
ISSN1535-9484
AutoresPatricia Pinheiro, Murad Ghanim, M. Alexander, Ana Rita Rebelo, Rogerio S. Santos, Benjamin C. Orsburn, Stewart M. Gray, Michelle Cilia,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoThe green peach aphid, Myzus persicae, is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops. The green peach aphid, Myzus persicae, is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops. Aphids are small insects that feed exclusively on the phloem sap of plants, and cause significant damage to agronomic crops. However, their major economic importance is that they are the most numerous vectors of plant viruses, such as the poleroviruses in the Luteoviridae, which we will refer to collectively as luteovirids in this manuscript. Luteovirids are single stranded, positive sense, nonenveloped RNA viruses that infect a range of economically important crops and weedy hosts. Luteovirids, including Potato leafroll virus (PLRV) 1The abbreviations used are: PLRV, potato leafroll virus; IAP, inoculation access period; DAS-ELISA, double antibody sandwich-ELISA; AAP, acquisition access period.1The abbreviations used are: PLRV, potato leafroll virus; IAP, inoculation access period; DAS-ELISA, double antibody sandwich-ELISA; AAP, acquisition access period., cause severe yield losses in agronomic crops around the world and are transmitted exclusively by aphids in a circulative manner. Circulative transmission requires a series of spatially and temporally regulated, largely unknown protein interactions with the virus structural capsid proteins (1.Tamborindeguy C. Bereman M.S. DeBlasio S. Igwe D. Smith D.M. White F. MacCoss M.J. Gray S.M. Cilia M. 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Controlling aphid vectors using pesticides is costly, and to be effective, information about vector phenology is necessary. Disrupting an aphid's ability to transmit a virus into or within a crop represents a different approach and a promising means by which to control virus spread (3.Gray S. Cilia M. Ghanim M. Circulative, "nonpropagative" virus transmission: an orchestra of virus-, insect-, and plant-derived instruments.Adv. Virus Res. 2014; 89: 141-199Crossref PubMed Scopus (104) Google Scholar, 4.Whitfield A.E. Falk B.W. Rotenberg D. Insect vector-mediated transmission of plant viruses.Virology. 2015; 479–480: 278-289Crossref PubMed Scopus (303) Google Scholar). Aphids acquire and transmit luteovirids as intact virions, not viral RNA, and there is no evidence to show that luteovirids replicate in their aphid vectors (3.Gray S. Cilia M. Ghanim M. Circulative, "nonpropagative" virus transmission: an orchestra of virus-, insect-, and plant-derived instruments.Adv. 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Ultrastructure of potato leaf phloem infected with Potato leafroll virus.Virology. 1980; 105: 379-392Crossref PubMed Scopus (61) Google Scholar), in aphid cells, virions are never observed free in the cytoplasm. The observation that virus-containing tubular vesicles connect to aphid cellular organelles supports hypothesis that the virus is transported intracellularly through the gut endomembrane system. Membrane-bound vesicles containing virions in gut cells of Myzus persicae and other aphid species have been observed to connect to lysosomes and lysosomal-like organelles (10.Garret A. Kerlan C. Thomas D. Ultrastructural study of acquisition and retention of potato leafroll luteovirus in the alimentary canal of its aphid vector, Myzus persicae.Sulz. Arch. Virol. 1996; 141: 1279-1292Crossref PubMed Scopus (31) Google Scholar, 13.Garret A. Kerlan C. Thomas D. 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Using organismal, biochemical, molecular, proteomic, and imaging approaches, we show that high levels of cysteine proteases at the cell membrane in aphids reared on turnip are indirectly responsible for the host-dependent change in the virus transmission phenotype in M. persicae. Parthenogenic reproducing colonies of the same clonal lineage (genetically identical individuals) of M. persicae Sultz (the green peach aphid) were maintained on caged physalis (Physalis floridana) or turnip (Brassica rapa) at 20 °C with an 18-hour photoperiod for a minimum of four months prior to the experiments and proteomics analyses. To test the host switch effect on PLRV transmission, aphids from both colonies, turnip (T-Myzus) and physalis (P-Myzus), were transferred to PLRV-infected hairy nightshade (Solanum sarrachoides, HNS) detached leaves for an acquisition access period (AAP) of 24 h, which is enough time for virus acquisition by aphids (Fig. 1) (42.Power A. Seaman A. Gray S. 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Virol. 2008; 89: 2037-2045Crossref PubMed Scopus (68) Google Scholar) and used as the source of virus. After that, 10 aphids were transferred to each healthy potato cv. Red Maria seedling (n = 10 plants) for an inoculation access period (IAP) of 48 h, as described (42.Power A. Seaman A. Gray S. Aphid transmission of Barley yellow dwarf virus - inoculation access periods and epidemiologic implications.Phytopathology. 1991; 81: 545-548Crossref Google Scholar, 43.Sadeghi E. Dedryver C.A. Gauthier J.P. Role of acquisition and inoculation time in the expression of clonal variation for BYDV-PAV transmission in the aphid species Rhopalosiphum padi.Plant Pathol. 1997; 46: 502-508Crossref Scopus (19) Google Scholar). Potato seedlings were treated with imidacloprid to eliminate aphids after the IAP. After three weeks, systemic infection of PLRV was detected in the recipient plants by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using α-PLRV antibodies (Agdia, Elkhart, IN). To test whether the impact of turnip feeding on PLRV transmission was transient, T-Myzus were transferred to physalis plants for three days and then transferred to PLRV-infected HNS for a 48 h AAP. Similarly, P-Myzus were transferred to turnip plants for three days and then fed to PLRV-infected HNS detached leaves for the same 48 h AAP. Five aphids were transferred to healthy potato seedlings for the transmission assay for a 48 h IAP, in 10 replicates per treatment. Three weeks later, the systemic PLRV infection in potato plants was detected by DAS-ELISA using α-PLRV antibodies. The proportion of plants infected with PLRV was compared with noninfected plants using the Chi square test. We measured the effect of each host plant on aphid reproduction and weight. Fourth instar nymphs of P-Myzus were transferred to either a turnip or a physalis plant, and after 24 h, nymphs that had molted to adults were transferred to a fresh turnip or physalis plant in four biological replicates to measure the host effect on aphid reproduction. Fifteen days later, the progeny was counted. The individual weight of adults was obtained by averaging the weight of 15–30 adults per replicate. Progeny counts and adult weights were analyzed by one-way ANOVA, and the means were compared using the Student's t test. P- and T-Myzus were fed on PLRV-infected HNS plants for a 24 h AAP. Aphids were then transferred to an artificial diet (45.Kim J.H. Jander G. Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate.Plant J. Cell Mol. Biol. 2007; 49: 1008-1019Crossref PubMed Scopus (269) Google Scholar) sandwiched between thinly-stretched parafilm membranes for gut clearing. Aphid cohorts were collected at 24 h or 72 h after the start of the AAP for PLRV quantification by digital droplet PCR (ddPCR), using the QX100 droplet digital PCR system (Bio-Rad, Hercules, CA). The ddPCR reaction for PLRV consisted of 10 μl of 2X ddPCR Evagreen SuperMix (Bio-Rad), 1 μl of combined primers at 10 μm each (PLRV CP qPCR F1 5′-CTAACAGAGTTCAGCCAGTGG-3′; PLRV CP qPCR R1 5′-TGTCCTTTGTAAACACGAATGTC-3′), 7 μl of dH2O and 2 μl of DNA diluted at 1:800 in a final volume of a 20 μl reaction. The entire 20 μl reaction was then loaded into a disposable DG8 droplet generator cartridge secured in the cartridge holder (Bio-Rad). A total of 70 μl of droplet generator oil for Evagreen (Bio-Rad) was also loaded into the disposable DG8 droplet generator cartridge. The cartridge holder was placed into the QX100 droplet generator (Bio-Rad) where droplets were generated. Droplets were then transferred to a 96-well plate (Eppendorf, Hamburg, Germany) and the plate was sealed with an easy pierce foil seal (Bio-Rad). PCR amplification was carried out on the Applied Biosystems 2720 Thermocycler. The thermocycling conditions started at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s and 60 °C for 1 min, 1 cycle at 4 °C for 5 min, 1 cycle at 90 °C for 5 min and ending at 12 °C. Following amplification, the plate was inserted into the droplet reader cassette and loaded into the droplet reader (Bio-Rad). The droplets were automatically read at a rate of 8 wells per 15 min. The ddPCR droplet data were analyzed using the QuantaSoft analysis software Version 1.7.4 2014 (Bio-Rad), which presents the target results as copies per μl of PCR mixture. The default settings to analyze the data were as follows: experiment set to ABS; SuperMix: QX200 ddPCR EvaGreen Super; Channel 1: FAM; and Channel 2: Vic/Hex. The digital droplet reader determined the number of copies per μl using the Poisson Distribution calculator. The number of copies obtained in two independent experiments were averaged and analyzed by One-Way ANOVA. We used gel-based separation and quantification of intact proteins, 2D-DIGE, to measure the relative quantification of protein expression between P-Myzus and T-Myzus. Three biological replicates of T- and P-Myzus (all life stages) were weighed and frozen at −80 °C in 50 ml BD-Falcon (Franklin Lakes, NJ) for protein extraction. Care was taken to remove all plant and soil debris from the aphids before freezing, so as not to contaminate the aphid protein samples. Proteins were extracted using a TCA-acetone protein precipitation protocol optimized for 2-D gel electrophoresis of aphid proteins (46.Cilia M. Fish T. Yang X. McLaughlin M. Thannhauser T.W. Gray S. A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins.J. Biomol. Tech. 2009; 20: 201-215PubMed Google Scholar). Protein samples were labeled with Cy3 or Cy5 according to the manufacturer's instructions (GE Healthcare; Piscataway, NJ). A Cy2 internal standard containing an equal amount of proteins from all the biological replicates was used for relative quantification by DIGE technology. Cy-dye labeled samples were grouped randomly during 2-D gel electrophoresis so that each gel contained a Cy3 and a Cy5 labeled sample, together with the Cy2-labeled, pooled internal standard. A dye swap was performed to control for any labeling bias. Analytical gels containing Cy dye-labeled samples were used for quantitative analysis and preparative gels containing nonlabeled samples were used for spot picking. A total of 50 μg of each Cy dye-labeled sample or 500 μg of nonlabeled protein were loaded onto immobilized pH gradient (IPG) strips (pH 4 to 7, 18 cm; GE Healthcare) during an overnight passive rehydration of the strips according to the manufacturer's specifications for the analytical and preparative gels, respectively. The first dimension was run on the IPGphor II (GE Healthcare) at 20 °C with the following settings: step 1: step and hold for 500 V, 1 h; step 2: gradient 1,000 V, 2 h; step 3: gradient 8000 V, 3 h, and step 4: step and hold 8000V until 34,000 V for a total focusing time o
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