The effect of mineral oil on stylet activities and potato virus Y transmission by aphids

Mineral oil sprayed onto potato virus Y (PVY) infected tobacco plants reduced acquisition of this potyvirus by Myzus persicae (Sulz.). Although the pre-penetration activities of aphids were longer on oil treated leaves, the inhibitory effect of the oil could not be attributed to differences in the duration of stylet penetration. Aphids were therefore made part of a DC circuit in order to investigate their stylet activities during penetration of PVY infected source plants and healthy test plants. Both acquisition and inoculation of the virus were reduced by the presence of oil on the plant surface, but these reductions could not be related to electrically recorded differences in plant penetration behaviour. In particular, stylet punctures of plant cell membranes were not reduced by mineral oil. Non-behavioural reasons are suggested to explain the mode of action of the oil.     

Polymer webs to prevent virus transmission by aphids in seed potatoes

Electrical registration and visual observation of penetration behaviour combined with ELISA-technique showed that laboratory-made webs of synthetic fibers were very promising to protect plants against virus infection by aphids. It was demonstrated that stylet penetration by aphids into potato leaves could be prevented by covering plants with these materials. The recent mass production of polypropylene sealed fiber fleeces and polyethylene nettings have made field experiments possible to determine their effect on aphid probing behaviour and virus transmission.Field experiments with potatoes showed that one particular fleece completely protected the plants against virus transmission (PVY and PLRV) and with a second fleece and a netting a small proportion of plants were infected. In uncovered plots up to 25% of plants became infected. A seed potato crop covered with polypropylene fleece which is inspected regularly for damage will be fully protected against virus transmission.

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Résumé Des enregistrements électriques et des observations visuelles du comportement de pénétration, combinés avec l'usage de la technique ELISA, ont montré que l'usage de toiles de fibres synthétiques élaborées au laboratoire était très prometteur pour la protection des plantes contre des infections virales transmises par des aphides. On a pu démontrer que la pénétration du stylet dans des feuilles de pomme de terre pouvait être empèchée en couvrant les plantes avec ces matériaux. La production en masse récente de nappes en fibres de polypropylene soudées et de filets de polyethylène a rendu possibles des expériences de terrain pour déterminer leur effet sur le comportement de sondage des pucerons et sur la transmission des virus.Des essais en plein champ avec des pommes de terre ont montré qu'un type de nappe en particulier protégeait complètement les plantes contre la transmission de virus (PVY en PLRV) et qu'avec un second type de nappe et un filet une petite proportion de plantes était infectée. Dans des parcelles non couvertes la proportion de plantes infectées s'élevait jusqu'à 25%. Une récolte de pommes de terre semencières couverte d'une nappe de polypropylène régulièrement inspectée afin de remédier aux détériorations éventuelles sera totalement protégée contre la transmission de virus.

     

The effect of pre-acquisition starvation on aphid transmission of potyviruses during observed and electrically recorded stylet penetrations

The effect of pre-acquisition starvation on stylet penetration behaviour by the aphidMyzus persicae (Sulz.) and the consequent non-persistent transmission of the potyviruses beet mosaic virus (BMV) and potato virus Y (PVY) were investigated. Visual observations indicated that starved aphids initiated penetrations earlier and penetrated for shorter periods than non-starved insects. Tethering with a fine gold wire did not affect these observations with either starved or non-starved aphids, but starvation caused increased PVY and BMV acquisition efficiency, regardless of tethering. Tethered aphids were then made part of an electrical circuit and their stylet activities investigated in detail. Electrically recorded aphids also acquired and inoculated both potyviruses more efficiently when starved, and these acquisitions and inoculations were associated with stylet punctures of plant cell membranes. However, starvation did not affect the occurrence of electrically recorded membrane puncture, suggesting that non-behavioural factors may contribute to the enhancement of virus transmission by pre-acquisition starvation.     

Xylem ingestion by winged aphids

When aphids and their host plant are incorporated in a DC electrical circuit, phloem and xylem ingestion register as separate waveforms of the electrical penetration graph (EPG) signal. Aphids are primarily phloem feeders; xylem ingestion is seldom reported but can be induced experimentally by fasting the insects in desiccating conditions. In experiments with the black bean aphid, Aphis fabae Scop., young winged (alate) and unwinged (apterous) virginoparous adults were collected from their natal host plants (broad bean, Vicia faba L.) and allowed 3-h continuous EPG-recorded access to V. faba seedlings. Several aphids (47% of both morphs) showed ingestion from phloem sieve elements. Alate aphids also showed frequent xylem ingestion (60% of individuals), but no apterous aphids exhibited this activity. The EPG technique involves attachment of a fine gold wire electrode to each insect, a process that may affect normal behaviour at the plant surface. However, when the technique was modified to monitor the stylet activities of freely-settled aphids, high levels of xylem ingestion by alates were also recorded. The results suggest that the developmental physiology of winged aphids somehow predisposes them to xylem ingestion, possibly as a result of dehydration during the teneral period. Alate aphids may reduce their weight by fasting before take-off, giving aerodynamic benefits, but making rehydration, via xylem uptake, a priority following plant contact.     

Tobacco plants transformed with the potato virus YN coat protein gene are protected against different PVY isolates and against aphid-mediated infection

Tobacco plant lines transformed with the coat protein (CP) gene of the tobacco veinal necrosis strain of potato virus Y (PVY^N), and previously shown to be protected against mechanical inoculation with the virus, have now been tested for specificity and protection against virus infection mediated by viruliferous aphids. To determine the specificity of virus protection, two transgenic tobacco lines, A30 and A80, were challenged with several isolates of distinct PVY strains (PVY^N, PVY^O and PVY^C) by mechanical inoculation. Clear levels of protection against the PVY^O-isolates tested were maintained in the transgenic plants, although these levels were slightly lower than the protection against the homologous PVY^N strain from which the CP gene was derived. Interestingly, no protection against mechanical virus inoculation with the Gladblaadje isolate of PVY^C could be observed. To assess the levels of protection against aphid-mediated virus infection, two transgenic plant lines, A30 and D25, showing respective levels of protection of 95 and 80% against mechanical virus inoculation, were challenged using PVY^N viruliferousMyzus persicae. Virus inoculation using six aphids per plant, resulted in similar levels of protection in both transgenic lines as found previously for mechanical inoculation. Protection was maintained in both lines, even when as many as 60 viruliferous aphids were used per plant in the inoculation experiments.     

Role of the methionine cycle in the temperature-sensitive responses of potato plants to potato virus Y

Plant–virus interactions are greatly influenced by environmental factors such as temperatures. In virus-infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in such regulatory effects remain largely uncharacterized. To provide more insight into the mechanisms whereby temperature regulates plant–virus interactions, we analysed changes in the proteome of potato cv. Chicago plants infected with potato virus Y (PVY) at normal (22 °C) and elevated temperature (28 °C), which is known to significantly increase plant susceptibility to the virus. One of the most intriguing findings is that the main enzymes of the methionine cycle (MTC) were down-regulated at the higher but not at normal temperatures. With good agreement, we found that higher temperature conditions triggered consistent and concerted changes in the level of MTC metabolites, suggesting that the enhanced susceptibility of potato plants to PVY at 28 °C may at least be partially orchestrated by the down-regulation of MTC enzymes and concomitant cycle perturbation. In line with this, foliar treatment of these plants with methionine restored accumulation of MTC metabolites and subverted the susceptibility to PVY at elevated temperature. These data are discussed in the context of the major function of the MTC in transmethylation processes.     

Stylet penetration activities linked to the acquisition and inoculation of Candidatus Liberibacter solanacearum by its vector tomato potato psyllid

The tomato potato psyllid (TPP), Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is the main vector of the bacterium Candidatus Liberibacter solanacearum (Lso), a major disease of solanaceous crops. Feeding of TPP is associated with Lso transmission. However, very little is known about the stylet penetration activities linked to acquisition and inoculation of Lso. The electrical penetration graph (EPG)‐DC system was used to monitor stylet penetration activities during acquisition and inoculation of Lso by individual TPP on tomato [Solanum lycopersicum L. (Solanaceae)]. Female TPP from Lso‐free and Lso‐infected colonies were used in acquisition and inoculation tests, respectively. In the acquisition tests, TPP were tested for Lso after EPG recording of their stylet penetration activities on Lso‐infected tomato shoots. In the inoculation tests, samples from the tomato plants on which the stylet penetration of Lso‐infected TPP had been recorded were tested for Lso infection. The relationships between qPCR results and the EPG waveforms (C, G, D, E1, and E2) representing the main stylet penetration activities performed by individual insects in inoculation and acquisition tests were investigated. Results confirmed that a single adult TPP is capable of infecting a plant with Lso. Our data suggest that acquisition of the bacteria occurs during phloem ingestion (E2), and inoculation is likely associated with salivation into the phloem sieve elements (E1). The durations of EPG parameters were not significantly different between Lso‐infected and Lso‐free TPP (later shown by qPCR) in acquisition tests. In inoculation tests, the durations of E1 or E2 recorded from TPP on Lso‐infected and Lso‐free plants that were later shown by qPCR were not significantly different. However, C was shorter on Lso‐infected plants than on Lso‐free plants, where TPP performed phloem activities. The minimum plant access period required for Lso transmission by a single TPP was estimated to be ca. 2 h, with an acquisition threshold of about 36 min.     

Resistance to potato virus Y in somatic hybrids between Solanum etuberosum and S. tuberosum x S. berthaultii hybrid

Somatic hybrids between a potato virus Y (PVY) resistant Solanum etuberosum clone and a susceptible diploid potato clone derived from a cross between S. tuberosum Gp. Tuberosum haploid US-W 730 and S. berthaultii were evaluated for resistance to PVY. All but one of the tested somatic hybrids were significantly more resistant than cultivars Atlantic and Katahdin. However, none was as resistant as the S. etuberosum parent. One hexaploid somatic hybrid, possibly the product of a triple-cell fusion involving one S. etuberosum protoplast and two haploid x S. berthaultii protoplasts, was as susceptible to PVY infection as the cultivars. Tetraploid progeny of the somatic hybrids, obtained from crosses with Gp. Tuberosum cultivars, were neither as resistant as the maternal somatic hybrid parent, nor as susceptible as the paternal cultivar parent. It appears that the introgression of PVY resistance from (1EBN) S. etuberosum into (4EBN) S. tuberosum (EBN-endosperm balance number) will be successful through the use of somatic hybridization and subsequent crosses of the somatic hybrids back to S. tuberosum.     

Determination of aphid transmission efficiencies for N, NTN and Wilga strains of Potato virus Y

Potato virus Y (PVY, genus Potyvirus, family Potyviridae) causes high economic losses worldwide, especially in the production of seed potatoes (Solanum tuberosum). PVY control systems rely on measuring virus pressure and vector pressure in the field. Calculation of the vector pressure is based on the relative efficiency factors (REFs) of aphid species. These REFs express the transmission efficiency of aphid species in relation to the transmission efficiency of Myzus persicae, the most efficient vector of PVY. In this paper, we report on the determination of aphids' relative transmission efficiency factors (REFs) for isolates of the PVY strains PVY^N, PVY^NTN and PVY^N-Wi. Biotype Mp2 of M. persicae was tested for its transmission efficiency for six PVY isolates (one PVY^N, three PVY^NTN and two PVY^N-Wi isolates) and showed comparable average transmission efficiencies for all isolates. The transmission rate of this biotype for the six PVY isolates was set to 1 and Mp2 was used as an internal control in transmission experiments to determine the REFs of three other biotypes of M. persicae and 16 other aphid species (three biotypes per species when available) for the six PVY isolates. Comparing the calculated REFs for PVY^N with the REFs reported in the previous century for PVY^N, we observe overall comparable REFs, except for Aphis fabae, Aphis spp., Hyperomyzus lactucae, Macrosiphum euphorbiae and Rhopalosiphum padi, which have a lower REF in our experiments, and Aphis frangulae and Phorodon humuli, which have now a higher REF. Comparing the new REFs found for the PVY^NTN strains with the new REFs for PVY^N, we observe that they are overall comparable, except for A. frangulae (0.17 compared with 0.53) and Schizaphis graminum (0.05 compared with 0.00). Comparing the REFs calculated for PVY^N-Wi with those calculated for PVY^N, we can observe six aphid species with higher REFs (Acyrthosiphon pisum, A. fabae, Aphis nasturtii, Aphis spp., P. humuli and R. padi). Only the species A. frangulae shows a lower REF for PVY^N-Wi compared with the transmission efficiency of PVY^N. Three aphid species (Aulacorthum solani, Myzus ascalonicus and S. graminum) for which no REF was determined earlier were found to be capable to transmit PVY and their REFs were determined.     

Stylet penetration by larvae of the greenhouse whitefly on cucumber

Probing behaviour of Trialeurodes vaporariorum (Westwood) larvae was monitored using the DC electrical penetration graph (EPG) technique on the host plant cucumber. EPGs were recorded for 16 h, simultaneously with honeydew excretion using a honeydew clock. Three waveforms were distinguished: a pathway waveform (C), and two phloem waveforms, one with a high (H), and one with a low frequency (L) signal. The C waveform mainly occurred in the crawler stage of the 1st instar larvae. EPGs recorded from larvae during and after moulting indicated that the process involves stylet withdrawal; hence the stylets of each new instar need to penetrate again from the leaf surface to the phloem.All sessile stages, from L1 to pre-pupa, spent almost their entire time in waveforms H and L. These waveforms alternated more frequently in the early instars than during the later ones, in which the H waveform became predominant. The H waveform was highly correlated with honeydew excretion and thus phloem sap ingestion. The L waveform was not related to honeydew excretion but EPGs indicated that the stylet tips remain in a sieve element during both waveforms. Periods of honeydew production demonstrated a delay of 30–40 min in relation to the onset and end of H and L waveforms. This delay is presumably related to the time needed for food passing through, or emptying of, the insect's gut. From the 1st instar to the pre-pupa, the frequency of excreted honeydew droplets decreased but their size increased, causing a net increase of the excretion rate.     

Electrical penetration graphs from Cicadulina mbila on maize, the fine structure of its stylet pathways and consequences for virus transmission efficiency

Five distinct electrical penetration graph waveforms characterising the feeding behaviour of the leafhopper Cicadulina mbila Naudé (Homoptera: Cicadellidae) on maize (Zea mays L.) were obtained using a DC based system. The waveforms were distinguished by spectral features and by statistical analysis of their median voltages, durations and time to first waveform recording. By changing the polarity of the system voltage and the level of the input resistor it was shown that the waveforms are mainly determined by the electromotive force (emf) component. Based on the correlation between waveforms and the fine structure of the stylet pathways observed by transmission electron microscopy, insect's activities have been associated with five waveforms: stylet pathway formation (waveform 1), active ingestion (waveform 2), putative stylet work (waveform 3), salivation (waveform 4) and passive ingestion (waveform 5). Like waveform E1 and E2 of aphids, waveforms 4 and 5 of C. mbila correspond to feeding activities in sieve tubes. However, unlike aphids which probe briefly in non-vascular cells, waveform 2 corresponds to active ingestion in cells, where the cell content is partially ingested and hence the organelles' integrity severely affected. These observations suggest that this specific feeding feature, typical of leafhoppers, determines their ability to acquire geminivirus virions located in the plant cell nucleus.     

Information‐theory‐based model selection for determining the main vector and period of transmission of Potato virus Y

Potato virus Y (PVY, genus Potyvirus, family Potyviridae) is transmitted non‐persistently by aphids. It causes major losses in potato production (Solanum tuberosum), especially following seed tuber‐borne infection of plants. To limit the risk of PVY infection, seed potato production is located preferably in regions where vector pressure is low. The northern‐most high‐grade seed potato production area (HG zone) of Europe is in Finland. The aim of this study was to determine the incidence of aphid species with documented ability to transmit PVY and to use a modelling approach to determine their relative importance as vectors of PVY in the HG zone of Finland. Winged aphids were caught from six to seven potato fields in each of three growing seasons (2007–09) using yellow pan traps that were examined twice a week. Identification of more than 30 000 individuals indicated that 37% of the aphids belonged to nine species reported to transmit PVY. Incidence of PVY in seed lots was low (0–5.6%) and the seasonal increase of PVY incidence was also low in the potato crops. No potato‐colonising aphids were found on the plants in any of the years. The seasonal increase in PVY incidence was modelled using aphid counts in traps, the relative vector efficiencies of the aphids, virus resistance of cultivars, and the initial infection rate of the seed tubers as explanatory variables in generalised linear mixed modelling. Akaike's information criterion was employed to find the best set of explanatory variables for PVY in harvested tubers. Results of this modelling approach showed that the incidence of seed‐borne PVY infection and the early‐season vector flights are the most important factors contributing to the incidence of PVY in the yield. Compared to models with data from all potential vector species, models containing data from Aphis fabae only showed a better model fit with regard to the incidence of PVY in the harvested tubers. The explanatory power of the models was lost when A. fabae was omitted from the vector data, suggesting that other species play a negligible role as vectors of PVY in the HG zone of Finland. Results can be used to devise appropriate strategies for enhanced control of PVY.     

Effects of a granulosis virus,andBacillus thuringiensis on life-table parameters of the potato tubermoth,Phthorimaea operculella

Experiments were conducted to evaluate the effect of 3 different concentrations of a granulosis virus (GV), and a standard application ofBacillus thuringiensis Berliner, on the life table parameters of the potato tubermothPhthorimaea operculella (Zeller) in the laboratory. Survival from egg to adult emergence was lowest (0.4%) and no reproduction occurred when larvae were fed on tubers treated withB. thuringiensis (i.e. 200 mg Thiricide/kg potatoes). Survival was 0.8, and 11.1% on tubers treated with 2.0, and 0.2 larval equivalents/kg potatoes (LE) of the GV, respectively, and was significantly (p<0.05) reduced compared to that for the lowest GV concentration (0.002 LE), and the control, i.e. 34.7, and 32.5% survival, respectively. However, generation time was not affected by the GV and fecundity was reduced only at the highest concentration. As a result, the intrinsic rate of increase (r_m) was significantly reduced at the highest, slightly reduced at the intermediate, and was not different for the lowest GV concentration as compared to the control, i.e. 0.0004, 0.0071, and 0.0100 compared to 0.0105, respectively. The implications for a potential biological control of the potato tubermoth in rustic potato stores are discussed.      

Transformation of homozygous diploid potato with an Agrobacterium tumefaciens binary vector system by adventitious shoot regeneration on leaf and stem segments

Transformed potato (Solanum tuberosum) plants were obtained from homozygous diploid potato by using a transformation procedure in combination with an adventitious shoot regeneration method. Leaf and stem explants were inoculated with an Agrobacterium tumefaciens strain which contained a binary vector (pVU 1011) carrying the neomycin phosphotransferase gene. Shoot regeneration most effectively on stem explants, occurred within six weeks directly from the explants without introducing a callus phase. A strong seasonal influence on transformation efficiencies was observed. Analysis of a number of randomly selected regenerated shoots for their ability to root and form shoots on kanamycin-containing medium shows that over 90% of the regenerated shoots obtained are transformed. In a number of shoots transformation was confirmed by a test for the presence and expression of the NPT-II gene.     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.     

Ribonuclease and proteinase activities in potato leaves immunized against Phytophthora infestans

Local treatment of potato plants with arachidonic acid caused a repeated increase of ribonuclease and proteinase activities in leaves that were directly sprayed with this inducer. Immunization caused no significant systemic changes in the enzyme activity of other potato leaves. However, leaves above the induction zone in the first days after the challenge were found to have a pronounced ribonuclease activity increase in comparison to that gradually rising with disease development in inoculated leaves from plants that were not induced. The proteolytic activity in the leaves of immunized plants after the challenge was decisively on a lower level than that in the leaves of noninduced plants subjected to inoculation.     

Azadirachta indica metabolites interfere with the host-endosymbiont relationship and inhibit the transmission of potato leafroll virus by Myzus persicae

The effects of neem (Azadirachta indica A. Juss) seed kernel extracts (NSKE) and azadirachtin on the ability of Myzus persicae (Sulz.) to transmit potato leafroll luteovirus (PLRV) was studied. Moreover, it was investigated whether treatments with these compounds would exert an effect on larval growth and mortality, and on the aphid intracellular symbionts. Endosymbiotic bacteria play an essential role in the performance of aphids, and in luteovirus transmission by aphids. NSKE and azadirachtin were offered to one-day-old M. persicae nymphs via a membrane feeding system. The neem metabolites displayed a 100% mortality at doses higher than 2560 ppm. At intermediate doses, ranging between 320 and 2560 ppm, larval growth and mortality were affected in a dose-dependent manner. The transmission of PLRV by M. persicae was inhibited by 55–90%. The endosymbiont population of the aphid was clearly affected by a treatment with neem metabolites as the release of their most abundant protein, Buchnera GroEL, into the haemocoel of the aphid was inhibited. Moreover, morphological aberrations on the bacterial endosymbionts were observed in aphids which fed on 2560 ppm of azadirachtin. At doses lower than 160 ppm of NSKE or azadirachtin, the endosymbiont population of M. persicae, and mortality, growth and feeding behaviour were similar to that of the untreated groups of aphids. However, PLRV transmission was still inhibited by 40–70%. The possible targets of the neem metabolites in the aphid are discussed.     

Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector

Summary The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%–2% of the newly synthesized protein.     

Enhancement of display efficiency in yeast display system by vector engineering and gene disruption

Vector engineering and gene disruption in host cells were attempted for the enhancement of α-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.