Aphid behavioral responses to virus‐infected plants are similar despite divergent fitness effects

We compared the settling preferences and reproductive potential of an oligophagous herbivore, the pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), in response to pea plants, Pisum sativum L. cv. ‘Aragorn’ (Fabaceae), infected with two persistently transmitted viruses, Pea enation mosaic virus (PEMV) and Bean leaf roll virus (BLRV), that differ in their distribution within an infected plant. Aphids preferentially oriented toward and settled on plants infected with PEMV or BLRV in comparison with sham‐inoculated plants (plants exposed to herbivory by uninfected aphids), but aphids did not discriminate between plants infected with the two viruses. Analysis of plant volatiles indicated that plants inoculated with either virus had significantly higher green leaf volatile‐to‐monoterpene ratios. Time until reproductive maturity was marginally influenced by plant infection status, with a trend toward earlier nymph production on infected plants. There were consistent age‐specific effects of plant infection status on aphid fecundity: reproduction was significantly enhanced for aphids on BLRV‐infected plants across most time intervals, though mean aphid fecundity did not differ between sham and PEMV‐infected plants. There was no clear pattern of age‐specific survivorship; however, mean aphid lifespan was reduced on plants infected with PEMV. Our results are consistent with predictions of the host manipulation hypothesis, extended to include plant viruses: non‐viruliferous A. pisum preferentially orient to virus‐infected host plants, potentially facilitating pathogen transmission. These studies extend the scope of the host manipulation hypothesis by demonstrating that divergent fitness effects on vectors arise relative to the mode of virus transmission.     

Transmission characteristics and some other properties of bean yellow vein-banding virus, and its association with pea enation

Bean yellow vein-banding virus (BYVBV) has been found occasionally in mixed infection with pea enation mosaic virus (PEMV) in spring-sown field beans (Vicia faba minor) in southern England. Glasshouse tests confirmed that, like PEMV, BYVBV is transmissible by manual inoculation and by aphids in the persistent manner. However, BYVBV can be transmitted by aphids only from plants that are also infected with a helper virus, usually PEMV. Thus after separation from PEMV by passage through Phaseolus vulgaris it was no longer aphid-transmissible. It became aphid-transmissible again only after re-mixing in plants with PEMV or with a substitute helper, bean leaf roll virus (BLRV). It was not transmitted by aphids that fed sequentially on plants singly infected with PEMV and BYVBV. Thus the interaction between BYVBV and PEMV (or BLRV) that enables BYVBV to be transmitted by aphids seems to occur only in doubly infected plants. However, it was not transmitted by aphids from plants doubly infected with BYVBV and broad bean wilt virus (BBWV). BYVBV and PEMV were transmitted more readily by Acyrthosiphon pisum than by Myzus persicae; neither virus was transmitted by Aphis fabae. Phenol extracts of BYVBV-infected leaves were more infective than phosphate buffer or bentonite-clarified extracts and were sometimes infective when diluted to 1/1000. The infectivity of BYVBV in phosphate buffer extracts of leaves singly infected with BYVBV, unlike that in extracts of leaves doubly infected with BYVBV and PEMV (or BLRV), was destroyed by treatment with organic solvents. BYVBV infected 11 of 28 plant species that were inoculated with phenol extracts; seven of the infected species were legumes. No transmission of BYVBV was detected through seed harvested from infected field bean plants. Isometric particles c. 30 nm in diameter were seen in extracts of plants doubly infected with BYVBV and PEMV but not in extracts of plants infected with BYVBV alone. Leaves of plants infected with BYVBV, alone or with PEMV, contained membrane-bound structures c. 50–90 nm in diameter associated with the tonoplast in cell vacuoles. These structures were not found in healthy leaves. BYVBV has several properties in common with other known aphid-borne viruses that are helper-dependent and transmitted in a persistent manner. Possibly, as suggested for some of them, aphid transmission of BYVBV depends on the coating of its nucleic acid with helper virus coat protein.     

Necrosis in field beans (Vicia faba) induced by interactions between bean leaf roll, pea early-browning and pea enation mosaic viruses

Mixed infections with two or three viruses - bean leaf roll (BLRV), pea early-browning (PEBV) and pea enation mosaic (PEMV) - were detected in plants showing leaf curling, stunting and necrosis in a crop of field beans grown for seed in 1980. In glasshouse tests, field bean plants infected with any one of these viruses showed no necrosis, and plants infected with PEBV and PEMV together showed symptoms of PEMV only. However, mixed infection with BLRV and PEMV almost invariably induced severe stunting and leaf necrosis, and infection with BLRV and PEBV often induced both leaf and stem necrosis and sometimes caused early death. Thus it seems that the necrotic symptoms seen in the field were induced by interactions between BLRV and the other viruses. No transmission of PEBV was detected through seed harvested from the crop, but up to 5% transmission was detected through seed from experimentally-infected plants. The infected seedlings were symptomless.     

Comparative transmission of bean leaf roll and pea enation mosaic viruses by aphids

Acyrthosiphon pisum was a more efficient vector than Myzus persicae of bean leaf roll virus (BLRV), but the two species transmitted pea enation mosaic virus (PEMV) equally well and much more often than Megoura viciae. M. viciae did not transmit BLRV, and Aphis fabae did not transmit BLRV or PEMV. BLRV and PEMV were transmitted more often by nymphs of A. pisum than by adult apterae or alatae that fed on infected plants only as adults, but both viruses were readily transmitted by adults that had developed on infected plants. The shortest time in which nymphs acquired BLRV was 2 h, and 50 % transmitted after an acquisition period of 4 days. Some nymphs acquired PEMV in 30 min and 50% in 8 h. The shortest time for inoculation of BLRV by adults was 15 min, but some transmitted PEMV in probes lasting less than 1 min. The median latent periods of BLRV and PEMV in aphids fed for 12 h on infected plants were, respectively, 105 and 44 h. Clones of A. pisum differed in their ability to transmit BLRV and PEMV, and efficiency in transmitting the two viruses seemed to be unrelated. Some aphids that fed successively on plants infected with each virus transmitted both viruses, and infectivity with one virus did not seem to affect transmission of the other.     

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

Complex interactions between a plant pathogen and insect parasitoid via the shared vector-host: consequences for host plant infection

Plant viruses modify the development of their aphid vectors by inducing physiological changes in the shared host plant. The performance of hymenopterous parasitoids exploiting these aphids can also be modified by the presence of the plant pathogen. We used laboratory and glasshouse microcosms containing beans (Vicia faba) as the host plant to examine the interactions between a plant virus (pea enation mosaic virus; PEMV) and a hymenopterous parasitoid (Aphidius ervi) that share the aphid vector/host Acyrthosiphon pisum. Neither PEMV-infection of V. faba, nor the carriage of PEMV virions by A. pisum, affected the growth or morphology of the aphid, or the oviposition behaviour and development of A. ervi. The presence of developing Aphidius ervi larvae within Acyrthosiphon pisum did not affect the ability of the aphids to transmit PEMV. However, by reducing their longevity, parasitism ultimately decreased the time viruliferous aphids were able to inoculate plants. In terms of virus dispersal, parasitized aphids exhibited more movement around experimental arenas than unparasitized controls, causing a slight increase in the proportion of beans infected with PEMV. Exposure to adult Aphidius ervi caused Acyrthosiphon pisum to rapidly drop off bean plants and disperse to new hosts, resulting in considerably higher plant infection rates (70%) than that seen in control arenas (25%). The results of this investigation demonstrate that when parasitoids are added to a plant-pathogen-vector system, benefits to the host plant due to reduced herbivore infestation must be balanced against the consequences of parasitoid-induced aphid dispersal and a subsequent increase in the level of plant infection.     

NMR profiling of transgenic peas

A high throughput proton nuclear magnetic resonance spectroscopy method for the metabolite fingerprinting of plants was applied to genetically modified peas (Pisum sativum) to determine whether biochemical changes, so called 'unintended effects', beyond those intended by incorporation of a transgene, were detectable. Multivariate analysis of 1H NMR (nuclear magnetic resonance) spectra obtained from uniformly grown glasshouse plants revealed differences between the transgenic and control group that exceeded the natural variation of the plants. When a larger data set of six related transgenic lines was analysed, including a null segregant in addition to the wild-type control, multivariate analysis showed that the distribution of metabolites in the transgenics was different from that of the null segregant. However, the profile obtained from the wild-type material was diverse in comparison with both the transgenics and the null segregant, suggesting that the primary cause of the observed differences was that the transformation process selects for a subset of individuals able to undergo the transformation and selection procedures, and that their descendants have a restricted variation in metabolite profile, rather than that the presence of the transgene itself generates these differences.     

Development and application of ELISA assays for the detection of two members of the family Luteoviridae infecting legumes: Pea enation mosaic virus (genus Enamovirus) and Bean leafroll virus (genus Luteovirus)

An antigen‐coated plate enzyme‐linked immunosorbent assay (ACP‐ELISA) method was developed and validated for the detection of Bean leafroll virus (BLRV) and Pea enation mosaic virus (PEMV), two of the important viral pathogens of several legume crops. The coat protein (CP) gene of each of the viruses was bacterially expressed as a fusion protein containing an N‐terminal hexa‐histidine tag and used as an antigen to produce antisera in rabbits. The antiserum to BLRV could detect the virus in leaf samples in up to 1:1000 dilution, and the PEMV antiserum detected the homologous virus in leaf samples of dilutions up to 1:6400. No serological cross‐reactivity was observed between anti‐BLRV and anti‐PEMV sera. The ACP‐ELISA assays were then used for estimating the prevalence of these two viruses in alfalfa, pea and vetch over a three‐state area in the US Pacific Northwest over a 2‐year period and virus incidence was mapped. Availability of rapid and sensitive ELISA assays facilitate virus disease mapping efforts and screening germplasm for virus resistance.     

Molecular and genetic analysis of transgenic rice plants expressing the maize ribosome-inactivating protein b-32 gene and the herbicide resistance bar gene

We have previously transformed rice (Oyrza sativa L.) with the maize ribosome-inactivating protein b-32 gene (Zmcrip3a) and the phosphinothricin resistance gene (bar). In the present study, Southern blot hybridization analysis of 56 primary fertile transformants resulted in distinct band patterns, indicating that all the transformants had been generated by independent integration events and 30% of them contained a single copy of the transgene. Segregation analysis of 15 R0 plants revealed that transgene was stably transmitted to their progenies and Southern blot band patterns of R1 progenies remained the same as the corresponding parents, suggesting that all the loci of multiple integration events are genetically linked. Also, in most of the lines, physical presence of the b-32 transgene co-segregated with the phosphinothricin- resistant phenotype, confirming that the transgene is behaving as a normal locus in the genome. However, some of R1 seedlings that contained multiple copies of the transgene became sensitive to phosphinothricin, indicating that its expression was silenced. Immunoblot analysis demonstrated that b-32 protein was produced and the levels of expression differed in different lines, estimated to be 0.5–1% of total soluble protein in the leaf tissues. In addition, the transgene-encoded protein was preferentially processed in germinating seeds and young leaves of R2 transgenic plants in a way similar to that in maize kernels, suggesting that the processing mechanism is conserved in the germination stage between rice and maize. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of Biolistic and Agrobacterium -mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium -mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG-CAM, into rice (BX)Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium -mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium -mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two-cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium -mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium -mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium -mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.     

Photosynthetic alterations of pea leaves infected systemically by pea enation mosaic virus: A coordinated decrease in efficiencies of CO2 assimilation and photosystem II photochemistry

We have investigated photosynthetic changes of fully expanded pea leaves infected systemically by pea enation mosaic virus (PEMV) that often attacks legumes particularly in northern temperate regions. A typical compatible virus–host interaction was monitored during 40 post-inoculation days (dpi). An initial PEMV-induced decrease in photosynthetic CO₂ assimilation was detected at 15 dpi, when the virus appeared in the measured leaves. This decrease was not induced by stomata closure and corresponded with a decrease in the efficiency of photosystem II photochemistry (Φ_PSII). Despite of a slight impairment of oxygen evolution at this stage, PSII function was not primarily responsible for the decrease in Φ_PSII. Chlorophyll fluorescence imaging revealed that Φ_PSII started to decrease from the leaf tip to the base. More pronounced symptoms of PEMV disease appeared at later stages, when a typical mosaic and enations appeared in the infected leaves and oxidative damage of cell membranes was detected. From 30 dpi, a degradation of photosynthetic pigments accelerated, stomata were closing and corresponding pronounced decline in CO₂ assimilation was observed. A concomitant photoprotective responses, i.e. an increase in non-photochemical quenching and accumulation of de-epoxidized xanthophylls, were also detected. Interestingly, alternative electron sinks in chloroplasts were not stimulated by PEMV infection, which is in contradiction to earlier reports dealing with virus-induced plant stresses. The presented results show that the PEMV-induced alterations in mature pea leaves accelerated leaf senescence during which a decrease in Φ_PSII took place in coordinated manner with an inhibition of CO₂ assimilation.     

Phase separation of a plant virus movement protein and cellular factors support virus-host interactions

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.     

Analysis and stability of the Respiratory Syncytial Virus antigen in a T<Subscript>3</Subscript> generation of transgenic tomato plants

The integration, expression, and stability of the Respiratory Syncytial Virus (RSV)-F protein was analyzed in a T₃ generation of transgenic cherry tomato, Solanum lycopersicum L. cv. Swifty Belle, plants. Expression of the RSV-F antigen, under the control of the fruit-specific promoter E-8, was investigated in T₃ plants derived from a transgenic line, identified as #120. Transgene integration of the RSV-F gene in the T₃ generation was initially determined by polymerase chain reaction (PCR). PCR analysis from line 120-7-2 revealed that all T₃ plants were homozygous for the transgene; whereas, line 120-6-4 showed segregation for the transgene. Enzyme-linked immunosorbent assay (ELISA) was used to quantify levels of RSV-F protein in these plants, and protein levels ranged from 0–22 μg/g of fresh weight, with an average of ~3 μg/g fresh weight. Southern blot analysis of the highest expressing plants revealed presence of a single copy of the RSV-F transgene in these plants.